A myopic perspective on the future of protein diagnostics

Abstract

Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.

Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

“…The site-directed attachment of exactly one oligo per Nb opens interesting possibilities where binders against different epitopes on the same protein molecule can be brought together by hybridization between their attached oligos for proximity ligation (PLA) or extension (PEA) assays. In this manner both efficiency and specificity of binding might be enhanced (22)(23). Immunoassays that require dual recognition by binders provide higher specificity and can reduce the background compared to single binder-assays by reducing risks of cross reactivity for irrelevant target molecules (24).…”

“…In this manner both efficiency and specificity of binding might be enhanced. [22][23] Immunoassays that require dual recognition by binders provide higher specificity and can reduce the background compared to single binder-assays by reducing risks of cross reactivity by the affinity reagents for irrelevant target molecules. 24 However, identifying non-competing pairs of binders remains a challenging task.…”

Antibody validation: a view from the mountains

“…A future scenario envisaged for antibody use is the possibility of analysing thousands of proteins in millions of samples of plasma and other biospecimens cheaply and accurately (Ulf Landegren, Uppsala). This will require new standards for several steps, including: pre-analytical collection, treatment and storage of samples (see above);development and selection of reagents (more rapid, more cheaply, new constructs);novel high throughput assay formats; andinexpensive readouts with absolute quantification. Implementation of such large-scale protein analyses will have a significant impact on disease prevention, diagnostics, drug development and selection, and ‘wellness’ monitoring (see also [20] for further discussion of these issues).…”

“…He argues for a number of remedies, including greater use of recombinant antibodies, more transparency on the part of (certain) producers and ultimately preparedness of individual users to carry out their own validations in their technique of choice. Ulf Landegren and colleagues [20] follow up with their thoughts on the importance of achieving detection specificity sufficient to measure very low concentrations of target proteins such as troponin and other leakage markers, and how this may be achieved by proper design of diagnostic assays. Considerations of specificity also permeate the other articles in this special issue.…”