Performance Control Strategies of One-Step Immuno-Chromatographic Assay System forSalmonella Typhimurium

Abstract

To attain optimum sensitivity (defined as the minimum detection limit) in an assay, an immuno-chromatographic assay system for Salmonella typhimurium (see Figure 1) were arranged at three different stages of: preparation of the respective system components, integration of the analytical parts, and trial test after fabrication. Primary approaches focused on the manufacture of two major components. In preparing the gold-antibody conjugate as tracer, important steps that may eventually affect the system performance were an appropriate blocking of residual surfaces of gold particles after conjugation and the subsequent separation of excess antibody used. The immobilized antibody on nitrocellulose membrane brought about variable results according to the storage time of the antibody solution before addition and also incubation conditions of the added solution onto the membrane. In the second stage, analytical elements were adapted for the construction of the system. Chemical composition of a medium used for elution as well as for the component production significantly altered the signal intensity. The concentrations of the both antibodies (conjugated and immobilized) as well as the time for antigen-antibody reaction (reflected by the volume of sample absorption pad) were the critical parameters that regulated the relative magnitude of the signal to the background obtained at zero dose of analyte. As the final step of control, the system fabricated was adjusted to a signal-to-noise ratio by two methods. Thermal treatment of the NC membrane with the immobilized antibody was effective to eliminate the background signal, while a detergent addition into the sample solution could be used to increase the signal density within a limited concentration range of analyte. Under the conditions optimized for sensitivity, the chromatographic system detected 2.5x10⁵ cells/mL or higher after an assay time of approximately 15 min.