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 …