A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.