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A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrPCWD, the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrPCWD using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrPCWD was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrPCWD concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrPCWD in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.