Comparison of antibody-antigen interactions on collagen measured by conventional immunological techniques and atomic force microscopy

Recep Avci, Mary Schweitzer, Robert D. Boyd, Jennifer Wittmeyer, Andrew Steeled, Jan Toporski, Iwona Beech, Fernando Teran Arce, Brenda Spangler, Kelly M. Cole, David S. McKay

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

We have developed a means of using atomic force microscopy (AFM) to repeatedly localize a small area of interest (4 × 4 μm 2) within a 0.5-cm 2 area on a heterogeneous sample, to obtain and localize high-resolution images and force measurements on nonideal samples (i.e., samples that better reflect actual biological systems, not prepared on atomically flat surfaces). We demonstrate the repeated localization and measurement of unbinding forces associated with antibody-antigen (ab-ag) interactions, by applying AFM in air and in liquid to visualize and measure pplyclonal ab-ag interactions, using chicken collagen as a model system. We demonstrate that molecular interactions, in the form of ab-ag complexes, can be visualized by AFM when secondary antibodies are conjugated to 20-nm colloidal gold particles. We then compare those results with established immunological techniques, to demonstrate broader application of AFM technology to other systems. Data from AFM studies are compared with results obtained using immunological methods traditionally employed to investigate ab-ag interactions, including enzyme-linked immunosorbent assay, immunoblotting, and in situ immunofluorescence. Finally, using functionalized AFM tips with a flexible tether [poly(ethylene glycol) 800] to which a derivatized antibody was attached, we analyzed force curve data to measure the unbinding force of collagen antibody from its antigen, obtaining a value of ∼90 ± 40 pN with a MatLab code written to automate the analyses of force curves obtained in force-volume mode, The methodology we developed for embedded collagen sections can be readily applied to the investigation of other receptor-ligand interactions.

Original languageEnglish (US)
Pages (from-to)11053-11063
Number of pages11
JournalLangmuir
Volume20
Issue number25
DOIs
StatePublished - Dec 7 2004
Externally publishedYes

ASJC Scopus subject areas

  • General Materials Science
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Spectroscopy
  • Electrochemistry

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