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Lateral flow assays (LFAs) are extensively used in qualitative detection because of their convenience, low cost, fast results, and ease of operation. However, the sample volume used in a lateral flow assay is usually determined experimentally. We test and find that the flow velocity is influenced by sample volume, using fluorescent microspheres as label particles, when analyte concentration is fixed in a sandwich LFA. A model is developed based on mass-action kinetics and advection-diffusion-reaction equation, combing the conjugate pad