, 2002). Expression in the Escherichia coli BL21 strain and purification was followed according to Ferreira et al. (2002). Salivary antigen was obtained according to da Silva Vaz Jr et al. (1994). Briefly, partially engorged females from the Porto Alegre strain were dissected in PBS and salivary glands
were separated from other organs and frozen at −70 °C. Salivary glands were macerated CHIR-99021 mouse and sonicated (Ultrasonicator Cole Parmer, 4710, 500 W, 4 and 20% duty cycle) in a solution containing Tris/HCl 10 mM pH 8.2, 1% deoxicolate, leupeptin (8 mg/ml), pepstatin A (1 mg/ml) and TPCK (0.1 mM) and centrifuged at 32,000 × g for 40 min at 4 °C. The soluble fraction (supernatant) was then collected and stored at −70 °C. Sera from six Bos taurus (Hereford) and eight B. indicus (Nelore) bovines from a farm in Pelotas (Brazil), within a region naturally infested with R. microplus, as well as the sera from non-infested
B. indicus animals (negative controls) Selleckchem MLN0128 were kindly provided by the Departamento de Veterinária Preventiva, at the Universidade Federal de Pelotas (Brazil). Additionally, the sera from three bovines (indicated as bovines 1, 2 and 3) submitted to twelve successive experimental infestations were the same described previously by Cruz et al. (2008). Briefly, the infestation regime consisted of six initial heavy infestations with 18,000 larvae (Bagé strain) followed by six light infestations with 800 larvae. All infestations were performed once a month and along the only back. rBmPRM was submitted to SDS–PAGE 10% (58 μg/cm) and transferred to the nitrocellulose membrane at 70 V for 1 h at 4 °C (Dunn, 1986). Nitrocellulose strips of 4 mm were blocked for 1 h at room temperature with blocking buffer (cow non-fat dry milk 5%–PBS). Prior to the overnight incubation at 4 °C with the membrane strips, all sera were diluted 1:50 in an E. coli
BL21 strain lysate expressing the pGEX-4T3 vector and incubated for 2 h at room temperature for removal of contaminating anti-vector and E. coli proteins reactive antibodies. As positive control an anti-rRmPRM hyperimmune serum (1:400) raised in bovine was used. Preparation of the E. coli BL21 strain lysate was performed according to Rott et al. (2000). After 3 washes with blocking buffer, the strips were incubated for 1 h with anti-bovine IgG peroxidase conjugate (Sigma), diluted 1:6000 in blocking buffer. The strips were then washed three times with PBS, and the development buffer (5 mg 3,3-diaminobenzidine in 30 ml PBS plus 150 μl H2O2 30% and 100 μl CoCl2 1%) was added. Microtitration plates were incubated overnight at 4 °C with 0.5 μg of rBmPRM or 1 μg salivary gland protein extract diluted in 50 mM carbonate/bicarbonate buffer pH 9.6 per well. Plates were washed three times with blocking buffer, blocked for 1 h with blocking buffer at 37 °C, and then incubated with bovine sera diluted 1:50 in blocking buffer for 1 h at 37 °C.