Immobilization strategies for single-chain antibody microarrays.

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.

Engineering an ultra-stable affinity reagent based on Top7.

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.