title: A modular design of low-background bioassays based on a high-affinity molecular pair barstar:barnase.
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.
authors: MQ Biofocus Research Centre, Macquarie University, North Ryde, NSW, Australia.
authors: Varun K A
authors: VK
authors: Sreenivasan
authors: Timothy A
authors: TA
authors: Kelf
authors: Ekaterina A
authors: EA
authors: Grebenik
authors: Oleg A
authors: OA
authors: Stremovskiy
authors: Jana M
authors: JM
authors: Say
authors: James R
authors: JR
authors: Rabeau
authors: Andrei V
authors: AV
authors: Zvyagin
authors: Sergey M
authors: SM
authors: Deyev
mesh_terms: D000906:Antibodies / Q000235:genetics; D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008856:Microscopy, Fluorescence; D040081:Protein Array Analysis / Q000379:methods; D040901:Proteomics / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D012260:Ribonucleases / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism
chemical_list: D000906:Antibodies; D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D018719:Erb-b2 Receptor Tyrosine Kinases; D012260:Ribonucleases; D019809:Streptavidin; C057438:barstar protein, Bacillus amyloliquefaciens; C508053:ERBB2 protein, human; C019760:Bacillus amyloliquefaciens ribonuclease