Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen

Tucci, Paula; Estevez, Verónica; Becco, Lorena; Cabrera-Cabrera, Florencia; Grotiuz, Germán; Reolon, Eduardo; Marín, Mónica

Rabbit antibodies against recombinant LKT, produced and purified by our work group, were used to identify LKT in culture supernatants. Briefly, membranes were incubated for 1 h at room temperature with anti-LKT antibodies at final concentration of 17.5 μg/mL in PBS 1X pH 7.4, 5% low fat milk, with gentle agitation. For antigen-antibody detection membranes were incubated for 1 h at room temperature with a 1:5000 dilution of anti-rabbit IgG (whole molecule)alkaline phosphatase antibody produced in goat (Sigma) in PBS 1X pH 7.4, 5% low fat milk. The intensity of resulting bands was quantified using a Molecular Imaging Software (Carestream Health Inc).