RESEARCH ARTICLE Immobilization strategies for single-chain antibody microarrays Shannon L. Seurynck-Servoss 1 , Cheryl L. Baird 1 , Keith D. Miller 2 , Noah B. Pefaur 2 , Rachel M. Gonzalez 1 , David O. Apiyo 1 , Heather E. Engelmann 1 , Sudhir Srivastava 3 , Jacob Kagan 3 , Karin D. Rodland 1 and Richard C. Zangar 1 1 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA 2 Biotechnology Group, Pacific Northwest National Laboratory, Richland, WA, USA 3 Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, NIH, Bethesda, MD, USA 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 anti- epitope 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 sug- gest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems. Received: November 9, 2007 Revised: February 8, 2008 Accepted: February 11, 2008 Keywords: Antibody microarray / ELISA / Single-chain antibodies Proteomics 2008, 8, 2199–2210 2199 1 Introduction Recent studies suggest that a pattern of proteins, rather than a single protein, can better diagnose complex diseases such as cancer [1–4]. Current clinical techniques cannot efficiently analyze multiple proteins with limited sample volumes, and therefore may have difficulties working with the small volumes that are available from large archived clinical stud- ies. ELISA microarrays are capable of multiplexing up to 50 assays within 1 array, greatly reducing the time and total Correspondence: Dr. Richard C. Zangar, Pacific Northwest National Laboratory, 790 Sixth Street, Richland, WA 99354, USA E-mail: richard.zangar@pnl.gov Fax: 11-509-376-6767 Abbreviations: EGF, epidermal growth factor; FACS, fluorescent activated cell sorting; His, histidine; HSV, herpes simplex virus; IL18, interleukin 18; PBS-T, 0.05% Tween 20 in PBS; PSA, pros- tate-specific antigen; scFv, single-chain antibodies; TRX, thiore- doxin; TX, toxin X DOI 10.1002/pmic.200701036  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com