Immunochemical characterization of antibodies labeled with vinylsulphonic triazinic dyes

FRIEDRICH, ADRIÁN; LEONI, JULIANA; FERRARI, ALEJANDRO

Buenos Aires

Congreso; 1er Congreso Franco ? Argentino de Inmunología (FAIC); 2010

For several decades, colloidal gold has been the first choice for labeling antibodies to be used in Point Of Care assays. These assays, like dot blot and immunochromatography (IC), require adequate sensitivity and low costs. In this context, triazinic dyes have been proposed as a new generation of protein dyes. It has been previously demonstrated that immunoglobulins (Igs) can be labeled with dichlorotriazones (Procion MX series), and that these Abs can be succesfully used for IC. Nevertheless, neither the sensitivity that can be achieved with these systems nor the stability of the label have been stablished. For the reasons mentioned above, we have optimized the labeling conditions of rabbit Igs raised against human IgG, we have explored the properties of Igs labeled with a vinylsulphonic triazinic dye (Remazol Brilliant Violet 5R), and characterized their properties within a capture dot blot assay.The conditions for optimum labeling have been assessed using a variable-size simplex strategy. Igs were best labeled at pH 10.9, 37 ºC, during an incubation of 132 min at a molar ratio of 264:1 (dye:Ig). When these antibodies were used in a capture dot blot assay, linearity was demonstrated and the sensitivity was high enough to detect as low as 40 ng of antigen in the reaction volume (400 ul).The results presented here show that this family of dyes is suitable for Ig labeling. These dyes provide extraordinary sensitivity, and are a promising replace of colloidal gold. Furthermore, these studies confirm that each subfamily of triazinic dyes requires specific labeling setup, as our optimum labeling conditions are not equal to those reported in literature