Two step lateral flow assay methods and devices

A two step lateral flow assay method for identifying specific IgE antibodies in a sample, comprising

• applying a blood sample to a sample port of a device, the device comprising
• a housing provided with a sample port, a buffer port upstream of the sample port, a result window downstream of the sample port, and a control window downstream of the result window,
• a buffer well upstream of the sample port and adapted to receive a quantity of liquid buffer applied through the buffer port,
• a lateral flow path within the housing, extending from the buffer well to the control window and comprising a lateral flow matrix,
• dried, labeled reagent adapted to bind IgE antibody, the labeled reagent being arranged on the lateral flow matrix downstream of the buffer well and upstream of the sample port, wherein the labeled reagent is adapted to be mobilized in the lateral flow matrix by liquid buffer passing from the buffer well along the lateral flow matrix, a plurality of IgE antigen species immobilized at respective positions on the lateral flow matrix at a first location visible through the result window, and
• a water-soluble mark located on the lateral flow matrix and visible through the control window prior to any addition of liquid to the device, wherein the water-soluble mark is dissolvable by a liquid sample that has passed through the immobilized IgE antigen species to provide a visual signal for addition of liquid buffer,
• allowing the sample to travel along the lateral flow matrix through the immobilized plurality of IgE antigen species, allowing any specific IgE antibodies in the sample to bind respective immobilized IgE antigen species, and allowing the sample to dissolve the water-soluble mark as the sample flows therethrough,
• applying a liquid buffer to the buffer port after the water-soluble mark is dissolved, allowing the liquid buffer to mobilize the labeled reagent, and allowing the immobilized liquid reagent to travel along the lateral flow matrix and bind with any IgE antibody bound to the immobilized IgE antigen species, and
• detecting any labeled reagent in the result window and relating the detected labeled reagent to a specific IgE antibody in the sample.