RAMPTM: A Rapid, Quantitative Whole Blood Immunochromatographic Platform for Point-of-Care Testing

Abstract

groups, which can react directly with both protein primary amine and sulfhydryl groups. Antibody monolayers. We used this activation chemistry to investigate whether the molecular packing of the IgG film formed on the water surface is maintained upon deposi-tion on solid supports. IgG molecules are known to form dense and stable monolayers on the water-air interface (3, 4) because of the pronounced hydrophobic regions on their solvent-exposed surface. The antibodies were conjugated with the dissociative Eu 3 label to calculate the surface density of the immobilized monolayers. The dependencies of the surface density and area per molecule on the surface pressure within the IgG monolayer are presented in Fig. 1. Both curves show pronounced inflections in the range of 20-30 mN/m. Although the curve for the area per molecule (curve b) is not properly a-A isotherm (because the total amount of molecules at the surface is not constant), it is obvious that the orientation of the molecules at the water-air interface changes with the surface pressure. These results demonstrate that antibody monolayers can be transferred from the water-air interface to the solid support and immobilized on it, simultaneously preserving their molecular organization. Receptor monolayers. We next performed a series of experiments to demonstrate that the antibody monolayer immobilized directly onto the siloxane polymer is comparable by surface density with the IgG layer that was specifically adsorbed onto the receptor sublayer. We first immobilized the Fc-binding receptors on the quartz supports modified with siloxane polymers. We used disso-ciative Eu 3 label and quartz crystal microbalance technique to measure the surface density of immobilized receptor monolayers. Both methods gave values between 5.8 and 8.9 pmol/cm 2. Ellipsometric measurements indicated that the thicknesses of the immobilized films were 4-5 nm (protein A and protein G) and 5-6 nm (protein A/G). These results suggest that the monolayers are densely packed and can prevent the nonspecific interactions of antibodies with the surface. Monoclonal anti-fluorescein (FITC) antibodies were then adsorbed to the receptor monolayers from the bulk solution, and the surfaces were blocked with bovine serum albumin. Low-molecular weight protein, adrenodoxin (M r 12 500), conjugated with FITC, was used as an antigen to analyze the binding capacity of the systems. Fluorescent microscopy was applied to collect fluorescence intensity profiles after the binding of FITC-adrenodoxin conjugate to anti-FITC antibodies oriented by receptor monolayers. The area of acquisition was 87 87 m 2. Five images at different places for every sample were acquired by charge-coupled device camera, and the integrated intensity was averaged. Our main results can be summarized as follows. Mono-layers formed from proteins A and G had approximately the same IgG binding ability, and the surface density of adsorbed antibodies was comparable to the IgG mono-layer formed at 20 mN/m. The chimeric receptor exhibits some advantage in binding capacity: the antibody film adsorbed onto the sublayer of protein A/G is comparable by density with the IgG monolayer formed at 30-40 mN/m. References 1. Jonsson U, Olofsson G, Malmqvist M, Ronnberg I. Method of silanization of surfaces. Thin Solid Films 1985;124:117-21. 2. Hoffmann PW, Stelzle M, Rabolt JF. Vapor phase self-assembly of fluorinated monolayers on silicon and germanium oxide. Langmuir 1997;13:1877-80. 3. Dubrovsky TB, Demcheva MV, Savitsky AP, Mantrova EYu, Yaropolov AI, et al. Fluorescent and phosphorescent study of Langmuir-Blodgett antibody films for application to immunosensors. The Rapid Analyte Measurement Platform (RAMP™) is an enabling immunodiagnostic platform for quantitative analysis of a wide variety of analytes. Consisting of an immunochromatographic strip in a disposable cartridge and a scanning dual wavelength fluorescence reader, RAMP takes advantage of the inherent simplicity of lateral flow immunochromatography while providing a quantitative measurement of analyte concentration. Using Fig. 1. Dependencies of the surface density (a) and the area per molecule (b) on surface pressure in IgG monolayers immobilized on silanized surfaces. The surface concentrations of antibodies were determined by using dissociative Eu 3 label.