Ligand assays: from electrophoresis to miniaturized microarrays

The analysis indicates that the attainment of high microspot assay sensitivity requires a detector capable of determining very low surface densities of labeled developing antibodies, the close packing of sensor-antibody molecules within the microspot— by minimizing background— contributing to the fulfillment of this requirement

Roger P Ekins


Key concepts

Scholarcy highlights

  • 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 methods, which rely on the use of labeled antibodies and high specific activity nonisotopic labels; 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
  • Many observers were initially skeptical of the possibility of developing ultrasensitive microspot ligand assays because the use of “vanishingly small” amounts of capture binding agent located on a small area contravenes widely accepted assay design rules stemming from the slope definition of sensitivity
  • Ligand assays have made a major impact on medicine and related fields in the past 40 years
  • Time-consuming and laborious, the technology was transformed in the 1980s by the emergence of noncompetitive labeled antibody methods, yielding higher sensitivities in much shorter times than the original techniques and opening the way to the development of the many automatic instruments that dominate the field at the present time
  • Miniaturized microarray technologies of the kind described in this article are suited to diagnostic applications because— only requiring small sample volumes —they do not merely constitute miniaturized versions of conventional assay methods and do not themselves rely on the micropumps, microchannels, micrometersized reaction compartments, and other micromachined structures embodied in many of the microanalytical devices currently under development. In consequence, do they rely on the measurement of submicroliter sample volumes of such minuscule size that assay sensitivities are severely reduced and large statistical variations are likely to be encountered in the numbers of molecules that are present in the measured sample
  • This prediction will only be fulfilled if they match current methods in regard to sensitivity, precision, and speed of performance while yielding substantial improvements in convenience and cost

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