Immobilization strategies for single-chain antibody microarrays

• Published in: Proteomics (Weinheim) Vol. 8; no. 11; pp. 2199 - 2210
• Main Authors: Seurynck-Servoss, Shannon L, Baird, Cheryl L, Miller, Keith D, Pefaur, Noah B, Gonzalez, Rachel M, Apiyo, David O, Engelmann, Heather E, Srivastava, Sudhir, Kagan, Jacob, Rodland, Karin D, Zangar, Richard C
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.06.2008; WILEY-VCH Verlag; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies - chemistry; Antibody microarray; Biological and medical sciences; Cell Separation; ELISA; Enzyme-Linked Immunosorbent Assay - instrumentation; Enzyme-Linked Immunosorbent Assay - methods; Epidermal Growth Factor - chemistry; Epitopes - chemistry; Fundamental and applied biological sciences. Psychology; Humans; Immunoglobulin Fragments - chemistry; Immunoglobulin G - chemistry; Immunoglobulin Variable Region - chemistry; Mice; Miscellaneous; Protein Array Analysis - methods; Proteins; Proteins - chemistry; Proteomics - methods; Single-chain antibodies; Thioredoxins - chemistry; Single-chain antibodies; Proteomics; Immobilization; Antibody microarray; ELISA assay; Single chain antibody; ELISA
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200701036

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 (R² = 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.