Sharp Transition in the Immunoimmobilization of E. coli O157:H7

Abstract

This work focuses on immobilization of living enterohemorrhagic Escherichia coli O157:H7 on a gold surface as a function of the concentration of antibody tethered to the surface in the physiological environment of the organisms. Experiments are conducted using antibodies raised against bacterial surface lipopolysaccharides (LPS) tethered to gold-coated silicon wafers at surface concentrations spanning a range from submonolayers of antibodies to full coverage, an estimated 1 antibody per ∼100 nm(2). A careful optimization of surface chemistry is conducted to obtain the most efficient tethering of the antibodies to the surface. The mechanism of immobilizing the bacteria is antibody-antigen interactions between the tethered antibodies on the surface and the bacterial surface LPS firmly attached to the bacteria. This type of attachment is known as immunoimmobilization. The experiments suggest no noticeable bacterial attachment until the surface antibody concentration reaches ∼70% of a full monolayer of coverage. Above this critical antibody density, a sharp increase in immunoimmobilized bacteria is observed as they populate nearly 80% to 100% of the available surface area, reaching ∼1.2 cells/10 μm(2). This sharp increase in population is tentatively explained in terms of the minimum number of antibody-antigen interactions required per bacterium to immobilize the cell. This critical number is estimated to be ∼6000-8000 antibodies per bacterium (having a 1 μm(2) footprint on the surface) under the assumption that a full monolayer of antibodies is about 1 antibody per ∼100 nm(2). However, the large majority of the 6000-8000 antibodies are not expected to participate in antibody-antigen interactions, in that the loose LPS in solution will saturate many of these antibodies before bacteria have a chance to interact with them. Furthermore, the geometric considerations will further restrict the majority of the active antibodies from interacting with the surface antigens of the cell, reducing its effective contact area with the antibodies considerably.

Surface Plasmon Resonance and Bending Loss-Based U-Shaped Plastic Optical Fiber Biosensors

“…The U-shaped POF probes were immobilized with rabbit anti- Escherichia coli polyclonal antibody (Bio-Rad, Hercules, CA, USA). The antibody immobilization protocol was adapted from [ 35 ], which is briefly described here.…”