title: Immobilization strategies for single-chain antibody microarrays.
abstract: 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.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Seurynck-Servoss
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Baird
authors.forename: Keith D
authors.initials: KD
authors.lastname: Miller
authors.forename: Noah B
authors.initials: NB
authors.lastname: Pefaur
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Gonzalez
authors.forename: David O
authors.initials: DO
authors.lastname: Apiyo
authors.forename: Heather E
authors.initials: HE
authors.lastname: Engelmann
authors.forename: Sudhir
authors.initials: S
authors.lastname: Srivastava
authors.forename: Jacob
authors.initials: J
authors.lastname: Kagan
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Richard C
authors.initials: RC
authors.lastname: Zangar
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods*; D004815:Epidermal Growth Factor / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013879:Thioredoxins / Q000737:chemistry
chemical_list: D000906:Antibodies; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011506:Proteins; D013879:Thioredoxins; D004815:Epidermal Growth Factor