Document Detail


Immobilization strategies for single-chain antibody microarrays.
MedLine Citation:
PMID:  18452230     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.
Authors:
Shannon L Seurynck-Servoss; Cheryl L Baird; Keith D Miller; Noah B Pefaur; Rachel M Gonzalez; David O Apiyo; Heather E Engelmann; Sudhir Srivastava; Jacob Kagan; Karin D Rodland; Richard C Zangar
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  Proteomics     Volume:  8     ISSN:  1615-9861     ISO Abbreviation:  Proteomics     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-06-10     Completed Date:  2008-09-09     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101092707     Medline TA:  Proteomics     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  2199-210     Citation Subset:  IM    
Affiliation:
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Antibodies / chemistry*
Cell Separation
Enzyme-Linked Immunosorbent Assay / instrumentation*,  methods*
Epidermal Growth Factor / chemistry
Epitopes / chemistry
Humans
Immunoglobulin Fragments / chemistry
Immunoglobulin G / chemistry
Immunoglobulin Variable Region / chemistry
Mice
Protein Array Analysis / methods
Proteins / chemistry
Proteomics / methods*
Thioredoxins / chemistry
Grant Support
ID/Acronym/Agency:
U01 CA117378/CA/NCI NIH HHS; Y1-CN-5014/CN/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies; 0/Epitopes; 0/Immunoglobulin Fragments; 0/Immunoglobulin G; 0/Immunoglobulin Variable Region; 0/Proteins; 52500-60-4/Thioredoxins; 62229-50-9/Epidermal Growth Factor

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


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