Lateral flow immunoassay device with separation membrane

A method of detecting an analyte of interest in a fluid sample, comprising:

• applying the fluid sample to a first flow path extending between a top surface and a bottom surface of a membrane configured to retain particles in the fluid sample;
• retaining particles in the fluid sample in the membrane:
• receiving the fluid sample in a second flow path spatially below and in fluid communication with the bottom surface of the membrane, the second flow path extending from a buffer receiving zone through a sample receiving zone to a capture zone downstream of the sample receiving zone, the sample receiving zone comprising a conjugate comprising a label and an agent configured to specifically bind to the analyte of interest, the capture zone comprising an immobilized capture agent specific to the analyte of interest;
• and
• adding a buffer to the buffer receiving zone such that the fluid sample received in the second flow path flow to the capture zone.

A lateral flow assay device for detecting an analyte of interest in a fluid sample comprising:

• a first flow path configured to receive the fluid sample, the first flow path extending between a top surface and a bottom surface of a membrane configured to retain particles in the fluid sample;
• a second flow path extending from a buffer receiving zone through a sample receiving zone to a capture zone downstream of the sample receiving zone, the sample receiving zone comprising a conjugate comprising a label and an agent configured to specifically bind to the analyte of interest, the capture zone comprising an immobilized capture agent specific to the analyte of interest, the second flow path spatially below and in fluid communication with the bottom surface of the membrane, the buffer receiving zone configured to receive a buffer that directs the fluid sample received through the bottom surface of the membrane along the second flow path to the capture zone;
• and
• a conjugate pad comprising the buffer receiving zone and the sample receiving zone, the bottom surface of the membrane configured to retain particles adhered to the top surface of the conjugate pad with double-sided adhesive.