Ligand assays: from electrophoresis to miniaturized microarrays

Abstract

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.

Solid supports for microarray immunoassays

“…Whereas the detection limit of DNA-microarrays enables the detection by fluorescence of high, medium and, in part, even low abundant transcripts (Kane et al, 2000), much better sensitivity is called for by the comparably large spectrum of proteins as well as the wider range of concentrations present within a given sample. Despite the fact that the theoretical detection limit of a microspot array with antibody monolayers was predicted to be a few femtograms or less (Ekins, 1998), it has been difficult until now to produce a detectable signal in the low picogram range even with artificial one-antibody one-antigen test systems, which avoid the background problems described above.…”

“…The history of miniaturized immunoassays started in the late 1980s, when Roger Ekins and coworkers created the first microspot multi-analyte immunoassay (Ekins et al, 1990a;Ekins, 1998). Stemming from this was the ambient analyte theory (Jackson and Ekins, 1986;Ekins, 1989Ekins, , 1994Ekins et al, 1990b), which describes microarray-based molecule interaction.…”

Assessment of the immunoglobulin E‐mediated immune response to milk‐specific proteins in allergic patients using microarrays

“…Most of the patients with CMA are young children and hence, the amount of sera available is very limited for the comparative studies and for studies that define patterns of allergen sensitization (CMA is usually caused by different allergen sensitizations). Miniaturized protein arrays open up new avenues in medicine because a minute spot area with immobilized samples provides greater sensitivity for the detection of molecular interactions compared with other binding assays [9]. This high-throughput and multiplexed protein array technology is proving to be attractive for the development of new immunoassays and diagnostic tools of antigen-caused pathologies (see [10] for a review).…”

Protein microarrays for genome‐wide posttranslational modification analysis

“…In a different study, Macbeath and Schreiber1 arrayed a panel of proteins on microarrays and provided a proof of concept for the ability to screen for protein–protein interactions, identify the substrates of protein kinases, and identify the protein targets of small molecules on solid surface. Commencing with Feinberg's3 description of a ‘microspot’ (see Box 1) and further developments by others,1,40–50 high‐throughput immunoassays that measure hundreds to thousands of mammalian or human proteins simultaneously have become the key for biomarker discovery and quantitative proteomic studies.…”