Roger Ekins

Development of microspot multi-analyte ratiometric immunoassay using dual fluorescent-labelled antibodies

Abstract Radioisotopes are progressively being replaced by non-isotopic labels as reagent markers for immunoassay purposes. The reasons for this development include an avoidance of the practical and environmental disadvantages associated with the use of radioisotopes and the need for higher assay sensitivity. A further stimulus is the increasing requirement to measure multiple analytes simultaneously in the same sample. This paper outlines the general principles underlying multi-analyte “microspot” immunoassay methodology. It relies on the measurement of the ratio of fluorescent signals from individual antibody “microspots” forming a microspot array on a suitable plastic surface. In principle, the methodology is capable of measuring 10 6 different substances in a sample volume of 100 μl.

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.

Single-Molecule ELISA

The need for assay methods of sufficient sensitivity to determine the low concentrations of hormones present in body fluids led to the original development of immunoassays and analogous “binding” (or “ligand”) assays in the late 1950s and early 1960s. These methods depend on the use of a binding agent (also commonly referred to by other terms such as “receptor,” “binding reagent,” and “analyte-specific reagent”), a substance used to recognize and bind the target analyte. Typical binding agents include antibodies, antigens, cell receptors, and serum binding proteins. Immunoassays still constitute the most widely used class of binding assays, although microarray-based nucleic acid assays, employing oligonucleotides as binding agents, are rapidly increasing in popularity. A principal objective in this field since the emergence of these assays has been to increase their sensitivities, especially in their application to certain analytes. Typifying such attempts, Rissin et al. (1) have recently reported a new approach to the further improvement of immunoassay sensitivities, claiming that with the use of an ELISA-type system, they were able to “detect serum proteins at subfemtomolar concentrations” and to increase the sensitivity of measurements “using a typical ELISA plate reader by a factor of about 68 000.” But before discussing the novel features of Rissin et al.s approach, we should briefly examine the concept of sensitivity and the meaning of the term “sensitive” to describe the performance of a binding assay—or indeed that of any measurement system. Many workers in this area, including Rissin et al., identify sensitivity with the lower limit of detection (LoD)3 of an assay. However, certain bodies, including the American Chemical Society and the International Union of Pure and Applied Chemistry, have formally defined sensitivity as the slope of the dose–response curve [or the response/dose (R/D) ratio—an intrinsically meaningless concept with which we strongly disagree (see …

Thyroid hormone regulates the expression of α-internexin in neurons in culture

Maternal hypothyroidism in the rat compromises &agr;-internexin (&agr;-IN) expression in early fetal brain. We have therefore examined whether 3,5,3′-triiodothyronine (T3) regulates &agr;-IN expression in fetal brain neurons in culture. Cells expressed transcripts encoding T3 nuclear receptor isoforms in a T3-independent manner. &agr;-IN protein abundance was increased in cultures treated with 0.1 and 1 nM T3 for 20 h (177 and 185% control, respectively) and in cultures treated with 1 nM T3 for 40 h (131% control). &agr;-IN transcript abundance was unaffected by T3 treatment. In conclusion, T3 at a physiological level, stimulates &agr;-IN protein, but not mRNA, levels in early differentiating neurons in culture. This supports the hypothesis that maternal thyroid hormone directly regulates early neuronal differentiation.