Comparison of antibody--antigen interactions on collagen measured by conventional immunological techniques and atomic force microscopy.
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.
Avci
Recep
R
Department of Physics, Montana State University, EPS 264, Bozeman, Montana 59715, USA. avci@physics.montana.edu
Schweitzer
Mary
M
Boyd
Robert D
RD
Wittmeyer
Jennifer
J
Steele
Andrew
A
Toporski
Jan
J
Beech
Iwona
I
Arce
Fernando Teran
FT
Spangler
Brenda
B
Cole
Kelly M
KM
McKay
David S
DS
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
D003094:Collagen