ABSTRACT We evaluated hemolyzed, bacterially contaminated, and Nobuto filter paper-derived serum, collected from 50 Rocky Mountain elk ( Cervus elaphus nelson ) in 2017 and 2019, divided into eight treatments to determine antibody retention. Serum was analyzed on Brucella abortus -specific fluorescence polarization assay utilizing plates and tubes. Reference titers and serostatus were compared to serum held at 22 C for 4, 8, 12, and 16 d; frozen clotted blood; blood with 2% and 10% elk rumen content (held for 8 d at 22 C); and serum eluted from Nobuto filter paper. Using Cohen's kappa test of agreement, plate assay serostatus agreement was substantial or outstanding in all treatments. Serostatus agreement was outstanding in all treatments utilizing tubes. The mean change in score (treatment minus reference) showed significant negative bias in serosuspect or seropositive animals in the frozen, 2% rumen, and 10% rumen treatments on the plate assay, and the day 16 and 10% rumen treatments on the tube assay, that could ultimately result in an animal being misclassified into a serosuspect or seronegative category. Serum eluted from Nobuto filter paper produced inconsistent results and is not recommended as an alternative to serum derived from blood. Although the potential for misclassification of animals with low titers exists, analyzing hemolyzed and bacterially contaminated serum from Brucella abortus nonendemic areas can increase sample size and the potential to detect seropositive animals.