Hrp conjugated monoclonal anti his tag antibodies

The purified recombinant protein was loaded onto 12% SDS-PAGE gels, fractionated and electrotransferred onto nitrocellulose membranes (Hybond ECL; GE Healthcare) on semidry equipment. A solution containing 10% non-fat dry milk in PBS 0.05% Tween 20 (PBS-T) was used to block unspecific sites in the membranes, which were then incubated with hamster serum for 1 h at room temperature. After extensive washing steps, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-hamster IgG (1:5000) in PBS-T 10% non-fat dry milk for 1 h. The transferred recombinant protein was also incubated with HRP-conjugated monoclonal anti-His tag antibodies (1:10,000) (Sigma, St. Louis, MO, USA). For the detection of human antibodies against epitopes contained in the chimeric protein in the leptospirosis serum samples, membranes were incubated with pooled MAT-positive, MAT-negative, or NHS samples (1:1000), and then, with HRP-conjugated anti-human IgG (1:10,000) or anti-human IgM (1:10,000) (Sigma). The negative control protein BSA was also included in these experiments. Detection was revealed using Super Signal West Dura Extended Duration Substrate (Thermo Fisher, Waltham, MA, USA).