Pbs freund s incomplete adjuvant

PD1063 antibodies were generated against a 14 amino acid peptide, GDYNKQKFRSIDLKC. The cysteine residue was added to the N-terminus to facilitate keyhole limpet hemocyanin adjuvant conjugation. Peptide synthesis and conjugation was conducted by GenScript USA Inc (New Jersey). The peptide was resuspended in 1× PBS/Freund's incomplete adjuvant (Sigma-Aldrich, Missouri) 50/50 for the first and five booster injections. Immunizations were conducted by the Comparative Pathology Laboratory at UC Davis. One hundred twenty-five micrograms of peptide was injected twice subcutaneously and twice intramuscularly, per injection date, into New Zealand White rabbits on a 10 day schedule. Test bleeds were taken after the fourth and fifth injections and the rabbits were terminally bled three weeks after the fifth injection. Serum was stored at −20°C until used. Indirect ELISA using synthesized non-conjugated peptide was performed to determine the bleed with the highest antibody titer and the optimal working dilution. Activity and specificity of the anti-PD1063 serum were evaluated using pre-immune serum in indirect ELISA and western blots. No non-specific adhesion of polyclonal, pre-immune rabbit antibodies was found (data not shown).

Purified HIS-gG3PD fusion protein was used to immunize intraperitoneally two BALB/c mice (Charles River Laboratories International, Inc., Wilmington, MA, USA) on days 0, 21, and 42 with 50 μg protein in 300 μl of emulsified 1:1 PBS/Freund’s complete adjuvant (Sigma–Aldrich; at day 0) or 1:1 PBS/Freund’s incomplete adjuvant (Sigma–Aldrich; at day 21) or without any adjuvant (at day 42). Blood was collected from the tail vein before initial immunization and after each boost. Sera fractions were assayed for specific antibody content.