Vivaspin tube

ANPs coated with FITC-conjugated PNA (ANP:PNA) were obtained through a noncovalent functionalisation of the particles by incubating a 1:1 ratio of ANPs and PNA at 4 °C for 1.5 h under stirring. The sample was concentrated using a 100 kDa Vivaspin^® tube (Sartorius), washed with deionised water and kept at −20 °C in a 20% glycerol solution, while the supernatant derived from the washing steps was stored for further analysis. The functionalisation with NGF and PNA was carried out by incubating a 1:1:1 ratio of ANPs, PNA and NGF (ANP:PNA:NGF), respectively, following the same protocol as above.

Fluorescent polyacrylamide nanoparticles were synthesised by optimising the radical polymerisation protocol developed by Rahimi and colleagues [38 (link)]. A total of 222 mg of NIPAM, 28.6 mg of AAm, 76 mg of AH and 262 μL of BIS were dissolved in 10 mL of deionised water previously purged with argon at room temperature and under stirring. Next, 11.5 μL of 10% SDS solution was added, and the solution was purged with argon for 30 min. Then, 1 mg of Alexa Fluor 488 or Alexa Fluor 594 dyes was dissolved to stop the argon flow, and 15.6 mg of APS and 20 μL of TEMED were added. Finally, deionised water was added to the final volume of 20 mL, and the reaction was carried out at room temperature for 3 h under continuous stirring in darkness. Dialysis was performed by transferring the sample into a 10 kDa cut-off membrane kept in deionised water under stirring and replaced with new water 4 times per hour. Finally, the sample was concentrated in a 30 kDa Vivaspin^® tube (Sartorius, Goettingen, Germany) by centrifuging at 3000× g until 1 mL of nanoparticle suspension was obtained, and then kept at 4 °C.

Immobilized Papain (Thermo Fisher Scientific, Waltham, USA) was used to digest PPI01 into its Fab and Fc fragments. PPI01 at 20 mg/mL was pipetted into 15 mL glass vial, the vial capped with the resin separator provided with the kit to remove all the air-liquid interface. The vial was gently rotated by a Sunlab rotator SU1100 for 5 h at 37 °C. An ÄKTA purifier 10 (GE Healthcare, Uppsala, Sweden) equipped with a Pierce Protein A chromatography cartridge (Thermo Fisher Scientific, Waltham, USA) (column volume, CV = 5 ml) was used to separate Fc (and undigested mAb) from the Fab fragments. The binding buffer was composed of 100 mM sodium phosphate with 150 mM NaCl at pH 7.2. The column was equilibrated with 2 CV of binding buffer with a flow of 2 ml/min. Fractions were collected in 15-ml PP tubes using a Frac 920 fraction collector (GE Healthcare, Uppsala, Sweden) capturing any unbound species e.g. Fab. The elution buffer (100 mM sodium phospate at pH 3) was kept at 100% over 7 CV. The eluting protein was collected in 15-ml PP tubes using the fraction collector, and was immediately neutralized with a 1 M sodium phosphate buffer at pH 8.5. Ultrafiltration was performed using Vivaspin® tubes with a 10 kDa MWCO PES membrane (Sartorius Stedim Biotech, Göttingen, Germany). Success of the purification was monitored by HP-SEC (see 3.4).

The excretory–secretory antigen of T. canis was prepared according to the method of de Savigny (1975). Larvae were cultivated in RPMI 1640 (Sigma-Aldrich, Hamburg, Germany) medium modified with 20 mM HEPES and l-glutamine, supplemented with penicillin/streptomycin (100 IU, 100 μg/ml) (Sigma-Aldrich, Hamburg, Germany). Larvae were maintained in sterile 25-ml tissue culture flasks (Falcon, Durham, USA) at a concentration of 10³ larvae/ml and incubated continually for a long term at 37°C under a 5% C0₂ and 95% atmosphere humidity. The culture medium was replaced at 5-day intervals after assessing the viability of larvae. Starting at the third week, the collected pooled medium (from 3 weeks) was concentrated in 3000 MWCO VIVASPIN tubes (Sartorius, Goettingen, Germany) at 4000 g for 100 min at 4°C. The protein concentration of the larval antigen was measured using the Bradford protein assay (Bio-Rad Laboratories, München, Germany).

HDL was isolated by a one-step density gradient ultracentrifugation method using long centrifuge tubes (16 × 76 mm; Beckman), as described^{34 (link)}. Briefly, the density-adjusted serum (1.24 g/mL with potassium bromide) was layered underneath a potassium bromide-density solution (1.063 g/mL). Samples were centrifuged at 330.000 x g for 6 h (centrifuge: Beckman Optima L-80 ultracentrifuge, rotor: Sorvall T-1270). Thereafter, the collected HDL was concentrated by Viva Spin Tubes (Sartorius, Vienna, Austria), desalted by gel filtration on Sephadex PD-10 columns (GE Healthcare, Munich, Germany) and either used directly or stored at −80 °C for further experiments.