Lateral flow
assay devices and methods for detecting a first member of a specific binding pair in a sample which comprises a plurality of
nonspecific binding pair members are adapted for
two step determinations. In one embodiment, a
two step lateral flow
assay method for identifying IgE antibodies in a sample comprises applying a sample to a sample port of a device, wherein the device is adapted to deliver the sample to a lateral flow matrix having a plurality of IgE
antigen species immobilized at respective positions at a first location The
two step method further comprises allowing the sample to travel along the lateral flow matrix through the immobilized plurality of IgE
antigen species to a second location downstream of the first location, applying liquid buffer to the lateral flow matrix to mobilize labeled
reagent which is adapted to bind anti-IgE
antibody and is dried on the lateral flow matrix at a location upstream of the delivery of the filtered sample to the lateral flow matrix, and allowing labeled
reagent mobilized by the liquid buffer to travel along the lateral flow matrix through the immobilized plurality of IgE
antigen species to a location downstream of the first location. Further embodiments comprise additional
lateral flow immunoassay devices and methods for identifying IgE antibodies in a sample.