Immobilized antibody orientation analysis using secondary ion mass spectrometry and fluorescence imaging of affinity-generated patterns

Anal Chem. 2010 Apr 1;82(7):2947-58. doi: 10.1021/ac902964q.


This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody "heads-up" and "tails-up" oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in "heads-up" regions, and ions from amino acids enriched in Fc domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Immobilized / chemistry*
  • Antibodies, Immobilized / immunology
  • Fluorescein / chemistry
  • Gold / chemistry
  • Hydrogels / chemistry
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fc Fragments / chemistry
  • Mice
  • Principal Component Analysis
  • Spectrometry, Mass, Secondary Ion / methods*
  • Staphylococcal Protein A / chemistry


  • Antibodies, Immobilized
  • Hydrogels
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Fc Fragments
  • Staphylococcal Protein A
  • Gold
  • Fluorescein