"Seeing and counting" individual antigens captured on a microarrayed spot with force-based atomic force microscopy.
The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.
Roy
Dhruvajyoti
D
Department of Chemistry, National Core Research Center for Systems Bio-Dynamics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea.
Kwon
Sung Hong
SH
Kwak
Ju-Won
JW
Park
Joon Won
JW
D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions; D006801:Humans; D008297:Male; D018625:Microscopy, Atomic Force / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D017430:Prostate-Specific Antigen