This present study was designed to evaluate four different Blastomyces dermatitidis antibody-antigen combinations (B5896 and T-58 antibodies and B5896 and WI-R antigens) for the detection of antigen in 36 urine specimens from dogs with blastomycosis using a standard indirect ELISA (STD) and a biotin-streptavidin ELISA (B-SA). The antigen detection sensitivity values ranged from 81% (B-SA: T-58 Ab + WI-R Ag) to 100% (STD and B-SA: B5896 Ab + WI-R Ag; B5896 Ab + B5896 Ag) with the antibody-antigen combinations in the two assays. Optimal detection was evidenced when the B5896 Ab was allowed to react with the urine specimens for 30 min at 37˚C and then placed in the B-SA ELISA plates containing the B5896 Ag. The greatest absorbance value obtained with this antibody-antigen combination was 0.903 (range of 0.596 -0.903) as compared to the control value of 1.246. The difference between the control absorbance and the test absorbance values was 0.343 which was considerably greater than the control-test values with the other combinations. This study thus showed that the results obtained in antigen detection assays are dependent upon the antibody used to react with the urine specimens as well as the antigen used in the enzyme immunoassay.