Immobilization strategies for single-chain antibody microarrays

Proteomics. 2008 Jun;8(11):2199-210. 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 (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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies / chemistry*
  • Cell Separation
  • Enzyme-Linked Immunosorbent Assay / instrumentation*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Epidermal Growth Factor / chemistry
  • Epitopes / chemistry
  • Humans
  • Immunoglobulin Fragments / chemistry
  • Immunoglobulin G / chemistry
  • Immunoglobulin Variable Region / chemistry
  • Mice
  • Protein Array Analysis / methods
  • Proteins / chemistry
  • Proteomics / methods*
  • Thioredoxins / chemistry


  • Antibodies
  • Epitopes
  • Immunoglobulin Fragments
  • Immunoglobulin G
  • Immunoglobulin Variable Region
  • Proteins
  • Thioredoxins
  • Epidermal Growth Factor