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Oriented Self-Assembling Protein Monolayers for Antibody Capture on Gold Surfaces


Deepan S H Shah, Orla Protein Technologies Ltd, Bioscience Centre, ICfL, Times Square, Newcastle upon Tyne, NE1 4EP


The immunoassay is a powerful tool in diagnostics; antibody technology provides exquisitely specific capture of biological markers for disease. In the last 15 years there has been a drive to transfer the benefits of the immunoassay onto the surfaces of advanced electronic biosensors. Traditional methods of antibody immobilisation such as adsorption and chemical coupling have some disadvantages that are magnified in the arena of ultrasensitive miniaturised electronic detection of antibody-antigen interaction (Table 1). In order to address some of these problems we have developed a method for creating oriented, stable, self-assembled monolayers of protein using engineered bacterial outer membrane proteins (omp) as scaffolds for fusion proteins [1,2]. The core technology requires the fusion of a protein of interest to a scaffold protein with self assembling properties. The fusion protein is assembled in a monolayer by a simple ‘apply-and-wash’ process. Gaps between the proteins are filled in with filler molecules such as PEG-thioalkanes, leaving only the protein of interest exposed (Figure 1). This method overcomes many of the problems associated with traditional methods of protein application to surfaces. The scaffold protein is highly stable with a melting temperature of 88°C. It is resistant to protease digestion and remains intact in SDS and extremes of pH. This basic technology was used to create a set of proteins for antibody immobilisation.


Table 1. Comparison of antibody immobilisation technologies. Property


Antibody orientation Non-specific binding Reproducibility


Complexity of manufacture Antibody density on surface Scale up for manufacture Antibody functionality


Proximity of antibody-antigen reaction to the surface


Adsorption No control


High Background Poor reproducibility Simple


Poor control Scale up problematic


Poor (~1-5% of immobilised antibody available for antigen)


Not controlled. Usually distant from surface.


Chemical Coupling


No control or only partial control Low to medium Reproducible


Involves complex chemistries Controllable


Scale up problematic


Reduced (25-50% available for antigen binding)


Large variations dependent upon chemistry and capture layers.


Orla Monolayer


Always in the correct orientation Non specific interactions are minimal Self assembly is highly reproducible Simple assembly from aqueous solution Controllable Easily scaled


Excellent (80-100% available for antigen)


The antigen binding site is ~20 nm from surface.


Self-assembling proteins for antibody capture


A set of IgG-binding proteins (Table 2) was generated by fusing tandem repeats of IgG-binding domains from Staphylococcus aureus protein A (SPA) [3], Streptococcus spp. Protein G (SPG) [4] and/or Peptostreptococcus Protein L (PPL) [5] to the Orla scaffold - an engineered variant of the E.coli outer membrane protein OmpA. SPA


and SPG bind to the Fc region of IgG whereas PPL binds to the κ-light chain of the variable domain. These proteins bind antibody in an orientation where the antigen- binding sites are exposed (Figure 2). The dimensions shown in Figure 2 were measured by polarised neutron reflection [6]. The total height of the capture layer and the antibody was close to 190 Å suggesting that the antibody is not completely upright as depicted but is partially tilted towards the surface. Consequently, the distance between the antigen binding site and the gold surface is only ~20nm and this is a great advantage for most types of biosensor.


Table 2. The IgG-binding protein set. Protein


Structure Orla18


Figure 1. Schematic diagram of protein monolayer formed using Orla technology. SPA-Omp 401


SPG-SPA-Omp 462 PPL-SPA-Omp 553 PPL-SPG-Omp 474


Amino acids Molecular Weight (kDa) 34.5


Orla85 SPG-Omp 404 Orla86 PPL-Omp 459 Orla87 Orla88 Orla89


44.0 44.5 50.6 51.1 60.6


Analysis of antibody-binding proteins by surface plasmon resonance


The IgG-binding properties of these proteins were examined using surface plasmon resonance (Biacore 2000 instrument, GE Healthcare). The binding of a rabbit polyclonal antibody and two of the common subclasses of mouse monoclonal antibodies were tested (Figure 3). It is apparent from these data that the different constructs provide a complex set of binding characteristics that may be exploited for different applications. In many cases the binding is extremely stable and only reversed by the acid regeneration.


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