Abstract:
:High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K(D)) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (K(D) ∼ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (K(D) ∼ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays.
journal_name
Proteomicsjournal_title
Proteomicsauthors
Sreenivasan VK,Kelf TA,Grebenik EA,Stremovskiy OA,Say JM,Rabeau JR,Zvyagin AV,Deyev SMdoi
10.1002/pmic.201200491subject
Has Abstractpub_date
2013-05-01 00:00:00pages
1437-43issue
9eissn
1615-9853issn
1615-9861journal_volume
13pub_type
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