Immobilization strategies for single-chain antibody microarrays

Journal Article · Sun Jun 01 04:00:00 EDT 2008 · Proteomics, 8(11):2199-2210

DOI:https://doi.org/10.1002/pmic.200701036· OSTI ID:935062

Seurynck-Servoss, Shannon L; Baird, Cheryl L; Miller, Keith D; Pefaur, Noah B; Gonzalez, Rachel M; Apiyo, David O; Engelmann, Heather E; Srinivastava, Sudhir; Kagan, Jacob; Rodland, Karin D; Zangar, Richard C

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays have great potential for validating biomarkers of disease. ELISA relies on robust affinity reagents that retain activity when immobilized or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional immunoglobin G (IgG). Unfortunately, scFv are typically less stabile than IgG and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv structural modifications to see if we could develop a more robust scFv reagent. Two promising strategies that emerged from these studies: 1) the precapture of epitope-tagged scFv using an anti-epitope antibody and 2) the direct printing of a thioredoxin/scFv fusion protein on glass slides. The use of either strategy improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the anti-epitope precapture method had a risk of reagent transfer. Using the direct printing method, we show that anti-PSA scFv are highly specific when tested against 21 different IgG-based assays. Finally, the scFv microarray PSA assay gave comparable results (R2 = 0.95) to a commercial 96-well ELISA when tested using serum samples. Overall, these results suggest that minor modifications of the scFv protein structure are sufficiently to produce reagents that are suitable for use in multiplex assay systems.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 935062
• Report Number(s): PNNL-SA-56848; 400412000
• Journal Information: Proteomics, 8(11):2199-2210, Journal Name: Proteomics, 8(11):2199-2210 Journal Issue: 11 Vol. 8
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
AFFINITY
ANTIBODIES
DETECTION
ENZYME IMMUNOASSAY
GLASS
MODIFICATIONS
PRODUCTION
PROTEIN STRUCTURE
PROTEINS
SENSITIVITY
STABILITY