Freund s incomplete adjuvant

In-vivo immunization of llama was performed following the protocol described by Pardon and co-workers with some modifications (Pardon et al., 2014 (link)). Briefly, 200 μg of rec-NadA was used for the first immunization followed by 5–weekly immunizations with 100 μg of the same protein (total 700 μg of protein used for immunization). For the first immunization, rec-NadA was mixed with Freund's complete adjuvant (1:1) (Sigma-Aldrich) and 1 mL of the mix was injected i/m in quadriceps femoris. Subsequent immunizations were performed using the emulsion of rec-NadA and Freund's incomplete adjuvant (1:1) (Statens serum institut, Copenhagen, Denmark). One week after the last immunization, 100 mL of blood was collected from the jugular vein in the presence of heparin (2500 IU) (Zentiva a.s., Czech Republic). Peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood using density gradient centrifugation in Histopaque medium (Sigma-Aldrich) as described in the manufacturer's instruction. RNA was isolated from the PBMC using the RNeasy mini kit (Qiagen) and reverse-transcribed with RevertAid (Thermo Scientific, Bratislava, Slovakia) following the manufacturer's instructions. sdAb-Not-R primer (Table 1) was used in the reverse transcription reaction.