A new enzyme immunoanalytical concept that can be used for point-of-care testing has been investigated. Enzyme as a tracer requires a separate reaction step for signal generation, which follows the completion of immune complex formation with analyte (e.g., Hepatitis B surface antigen) in a sample. This has been a major factor limiting its utilization within the laboratory. We carried out such sequential processes employing chromatographic analysis, using two crosswise-arranged membrane pads in vertical and horizontal directions. The vertically arranged pads were the same as those in the usual format for pregnancy testing, for instance, with the exception of the use of horseradish peroxidase (HRP) as tracer. By placing the horizontally arranged pads on each lateral side of the signal generation pad in the vertical arrangement, they were employed to supply substrate to the enzyme present in the immune complexes. The substrate flow was initiated after the antigen-antibody bindings to produce a signal, which was typically a color change in proportion to the analyte concentration. Under optimal conditions, the use of HRP labeling increased the detection capability of the assay approximately 30 times compared to that of gold colloids. Potential advantages of using the concept investigated are (1) provision of a rapid and simple immunoassay, (2) satisfaction of a clinical need for highly sensitive determination of analyte, and (3) utilization of relatively inexpensive, portable quantitation means.