Document Detail


Improvement of sensitivity and dynamic range in proximity ligation assays by asymmetric connector hybridization.
MedLine Citation:
PMID:  20704387     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The proximity ligation assay (PLA) is one of the most sensitive and simple protein assays developed to date, yet a major limitation is the relatively narrow dynamic range compared to other assays such as enzyme-linked immunosorbent assays. In this work, the dynamic range of PLA was improved by 2 orders of magnitude and the sensitivity was improved by a factor of 1.57. To accomplish this, asymmetric DNA hybridization was used to reduce the probability of target-independent, background ligation. An experimental model of the aptamer-target-connector complex (apt(A)-T-apt(B)-C(20,PLA)) in PLA was developed to study the effects of asymmetry in aptamer-connector hybridization. Connector base pairing was varied from the PLA standard of 20 total bases (C(20)) to an asymmetric combination with 15 total bases (C(15)). The results of this model suggested that weakening the affinity of one side of the connector to one aptamer would significantly reduce target-independent ligation (background) without greatly affecting target-dependent ligation (signal). These predictions were confirmed using PLA with asymmetric connectors for detection of human thrombin. This novel, asymmetric PLA approach should impact any previously developed PLA method (using aptamers or antibodies) by reducing target-independent ligation events, thus generally improving the sensitivity and dynamic range of the assay.
Authors:
Joonyul Kim; Jiaming Hu; Rebecca S Sollie; Christopher J Easley
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical chemistry     Volume:  82     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2010 Aug 
Date Detail:
Created Date:  2010-08-13     Completed Date:  2010-12-15     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6976-82     Citation Subset:  IM    
Affiliation:
Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University, Auburn, Alabama 36849, USA.
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MeSH Terms
Descriptor/Qualifier:
Aptamers, Nucleotide / chemistry*
Base Sequence
Humans
Models, Molecular
Nucleic Acid Hybridization / methods*
Oligonucleotides / chemistry*
Thrombin / analysis
Chemical
Reg. No./Substance:
0/Aptamers, Nucleotide; 0/Oligonucleotides; EC 3.4.21.5/Thrombin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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