Mouse IgG heterogeneous immunoassay development using electrochemical detection: Beads and buffers

An investigation involving enzyme-based sandwich immunoassays was done with respect to buffers, microbeads, and small volume immunoassay development. Mouse IgG was the analyte used for these studies. Alkaline phosphatase was incorporated as the enzyme label to convert p-aminophenyl phosphate (PAPP) to p-aminophenol (PAP) which could be detected electrochemically. Flow injection analysis with electrochemical detection was used to determine the amount of PAP produced. The current measured was proportional to the amount of analyte present in the immunoassay. A potential of 320 mV vs. Ag/AgCl was applied to a glassy carbon electrode for amperometric detection.; A buffer system (Tris, pH 9) had previously been established for the combination of alkaline phosphatase, PAPP, and PAP. This buffer is a compromise between optimal enzyme activity and PAP stability. A new buffer, one that includes glycine has been investigated with respect to enzyme activity, PAP stability, and effects on overall assay performance. Alkaline phosphatase activity was slightly lower in this new buffer system with kcat/K m values of 1.3 x 1010 M-1min -1 in Tris and 3.0 x 109 M-1 min-1 in Tris-gly. But, immunoassay results for mouse IgG in Tris-glycine buffer, showed that a 10 min increase in substrate incubation time with the enzyme lowered the detection limit due to a reduction in the background signal.; Small volume immunoassays are becoming increasingly necessary as sample sizes shrink. As volumes have become smaller and larger surface areas have been incorporated (due to decreased diffusional pathlengths), unusual enzyme kinetics have been observed. Using a 22 mul derivatized, fused silica capillary for the reaction vessel an attempt was made at understanding the kinetics by determining their source.; Finally, a mouse IgG preliminary immunoassay that used magnetic microbeads as the solid support has been developed. The assay used biotinylated primary antibody for attachment onto the streptavidin-coated microbeads. In addition to the actual immunoassay development, initial studies were started to reduce non-specific adsorption signals associated with the bead assays. Detection limits are currently 1 ng/ml using 106 microbeads.