An Internal Clock Reaction Used in a One-step Enzyme Immunochromatographic Assay of Theophylline in Whole Blood

We describe the development and performance of a second-generation enzyme immunochromatography method for visually quantifying theophylline in whole blood without the use of instrumentation. We have developed the novel concept of an internal chemical clock reaction to combine the capillary-migration and color-generation protocol of the two-step immunochromatographic assay into a single-step, simultaneous protocol. The two assay components are (a) chromatographic paper to which glucose oxidase (EC 1.1.3.4) and monoclonal antibody to theophylline have been immobilized, and (b) an enzyme reagent consisting of glucose, dicarboxidine, ascorbate, and theophylline-labeled horseradish peroxidase (EC 1.11.1.7). The ascorbate acts as an internal clock by inhibiting premature color formation until the ascorbate has been completely consumed in the peroxidase-mediated reaction. Color is then generated rapidly, producing a clearly visible front on the paper. Performance evaluations of the 20-min one-step assay show very good precision, analytical recovery, specificity, and accuracy. This simplified protocol is reliable and convenient for therapeutic drug monitoring in the physician's office.