Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement.
Atomic force microscope (AFM) was exploited to take picture of the molecular topology of C-reactive protein (CRP) in phosphate-buffered saline (PBS) solution. An explicit molecular image of CRP demonstrated a pentagonal structure composed of five subunits. Dimensions of the doughnut-shaped CRP molecule measured by AFM were about 25 nm in outside diameter and 10 nm in central pore diameter, and the height of CRP molecule was about 4 nm which was comparable to the value determined by X-ray crystallography. Bis(N-succinimido)-11,11'-dithiobis (undecyl succinate) (DSNHS) was synthesized for use as a linker for immobilizing anti-CRP antibody (anti-CRP) onto the gold surface of a surface plasmon resonance (SPR) sensor chip. DSNHS formed self-assembled monolayer (SAM) on the gold surface. By use of an AFM tip, a pattern of ditch was engraved within the SAM of DSNHS, and anti-CRP was immobilized on the engraved SAM through replacement of N-hydroxysuccinimide group on the outside surface of DSNHS by the amine group of anti-CRP. Formation of CRP/anti-CRP complex on the gold surface of SPR sensor chip was clearly demonstrated by measuring SPR angle shift. A consecutive series of SAM, SAM/anti-CRP, and SAM/anti-CRP/CRP complexes was generated on a SPR sensor chip, and the changes in depth of the ditch were monitored by taking AFM images of the complexes. Comparative analysis of the depth differences indicates that binding of CRP to anti-CRP occurs in a planar mode.
Lee
Soo-Keun
SK
Department of Biotechnology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 704-230, Republic of Korea.
Kim
Hyun-Chul
HC
Cho
Sang-Joon
SJ
Jeong
Sang Won
SW
Jeon
Won Bae
WB
D000906:Antibodies / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D002097:C-Reactive Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods
D000906:Antibodies; D002097:C-Reactive Protein