title: Comparison of antibody--antigen interactions on collagen measured by conventional immunological techniques and atomic force microscopy.
abstract: We have developed a means of using atomic force microscopy (AFM) to repeatedly localize a small area of interest (4 x 4 microm(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 polyclonal 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 approximately 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.
authors.affiliation: Department of Physics, Montana State University, EPS 264, Bozeman, Montana 59715, USA. avci@physics.montana.edu
authors.forename: Recep
authors.initials: R
authors.lastname: Avci
authors.forename: Mary
authors.initials: M
authors.lastname: Schweitzer
authors.forename: Robert D
authors.initials: RD
authors.lastname: Boyd
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wittmeyer
authors.forename: Andrew
authors.initials: A
authors.lastname: Steele
authors.forename: Jan
authors.initials: J
authors.lastname: Toporski
authors.forename: Iwona
authors.initials: I
authors.lastname: Beech
authors.forename: Fernando Teran
authors.initials: FT
authors.lastname: Arce
authors.forename: Brenda
authors.initials: B
authors.lastname: Spangler
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Cole
authors.forename: David S
authors.initials: DS
authors.lastname: McKay
mesh_terms: D000388:Air; D000818:Animals; D000937:Antigen-Antibody Reactions / Q000276:immunology / Q000502:physiology*; D002645:Chickens; D003094:Collagen / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D007158:Immunologic Techniques; D018625:Microscopy, Atomic Force / Q000379:methods*; D010316:Particle Size; D013499:Surface Properties
chemical_list: D003094:Collagen