Montanide isa 50 5 adjuvant

For each recombinant tick protein and total ISE6 tick cell proteins, two New Zealand white rabbits (Oryctulagus cuniculus) were subcutaneously injected at weeks 0, 4, and 6 with 50 μg protein in 0.4 ml Montanide ISA 50 V adjuvant (Seppic, Paris, France). Blood was collected before injection and 2 weeks after the last immunization to prepare pre-immune and immune sera, respectively. Serum aliquots were kept at 4°C for immediate use or at −20°C for long-term storage. The IgG were purified from serum samples using the Montage antibody purification kit and spin columns with PROSEP-A media (Millipore, Billerica, MA, USA) following the manufacturer's recommendations.

Rabbit antiserum against rPaR1 was obtained by immunizing 2 months-old New Zealand rabbits subcutaneously (s.c.) with 100 μg of purified rPaR1 protein emulsified with 50% (vol/vol) Montanide ISA 50 V adjuvant (Seppic) and boosted twice s.c. with 100 μg of rPaR1 at two weekly intervals. The rabbit sera were collected 7 days after the third immunization.

For each tick protein, three New Zealand white rabbits (Oryctulagus cuniculus) were subcutaneously injected at weeks 0, 3 and 6 with 50 μg protein in 0.5 ml Montanide ISA 50 V adjuvant (Seppic, Paris, France). Blood was collected before injection and two weeks after the last immunization to prepare preimmune and immune sera, respectively. Serum aliquots were kept at 4°C for immediate use or at −20°C for long-term storage. IgGs were purified from serum samples using the Montage Antibody purification kit and spin columns with PROSEP-A Media (Millipore, MA, USA) following the manufacturer’s recommendations.