title: Highly efficient binding of paramagnetic beads bioconjugated with 100,000 or more antibodies to protein-coated surfaces.
abstract: We report here the first kinetic characterization of 1 μm diameter superparamagnetic particles (MP) decorated with over 100,000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab(2)) exhibit irreversible binding with 100-fold increased association rates compared to free antibodies. The estimated upper limit for the dissociation constant of MP-Ab(2) from the SPR sensor surface is 5 fM, compared to 3-8 nM for the free antibodies. These results are explained by up to 2000 interactions of MP-Ab(2) with protein-decorated surfaces. Findings are consistent with highly efficient capture of protein antigens in solution by the MP-Ab(2) and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range. Aggregation of these particles on the SPR chip, probably due to residual magnetic microdomains in the particles, also contributes to ultrasensitive detection and may also help drive the irreversible binding.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, 06269, United States.
authors.forename: Vigneshwaran
authors.initials: V
authors.lastname: Mani
authors.forename: Dhanuka P
authors.initials: DP
authors.lastname: Wasalathanthri
authors.forename: Amit A
authors.initials: AA
authors.lastname: Joshi
authors.forename: Challa V
authors.initials: CV
authors.lastname: Kumar
authors.forename: James F
authors.initials: JF
authors.lastname: Rusling
mesh_terms: D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D015374:Biosensing Techniques; D006046:Gold / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000276:immunology / Q000378:metabolism*; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism*; D007610:Kallikreins / Q000276:immunology / Q000378:metabolism*; D058185:Magnetite Nanoparticles; D017430:Prostate-Specific Antigen / Q000276:immunology / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D055767:Immobilized Proteins; D015850:Interleukin-6; D058185:Magnetite Nanoparticles; D006046:Gold; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen