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Currently no rapid immunoassays are developed to identify the species content of fat tissue in mixtures. We report a simple protocol enabling the effective detection of bovine fat in highly processed materials using a lateral flow (LF) immunoassay which targets a ruminant-specific muscle protein. A portion (50 gm) of muscle-free fat samples was rendered to separate the molten fat from the proteinaceous residue, then soluble proteins were extracted from the solid residue with 0.5 mol/L NaCl for the LF analysis. The assay could detect 2% bovine fat-in-pork fat, 1% bovine fat-in-porcine meat-and-bone meal, and 0.5% bovine fat-in-soy meal mixtures. Rendered bovine fat could be detected up to 213 degrees C. These results demonstrate that low levels of bovine fat tissue can be detected in processed materials using an immunoassay based on the presence of the muscle protein which serves as a species marker in the fat tissue.