Freund complete adjuvant

Two 2-month-old rabbits were immunized with 0.2 mg rEtCDPK4 emulsified in Freund Complete Adjuvant (Sigma) by intraperitoneal injection. Rabbits were boosted three times with Freund’s incomplete adjuvant at 2-week intervals. Eight days after final immunization, polyclonal antibody serum was separated from two rabbits blood.
rEtCDPK4 was resolved by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bellerica, MA, USA). Western blots were performed according to standard procedures by using rabbit anti-merozoite protein sera (1:500) previously obtained in our lab or anti-His-tag monoclonal antibody (1:2000). Native rabbit IgG (1:1000) was the negative control. IRDye 800CW goat anti-rabbit IgG (LI-COR, Lincoln, NE, USA) and IRDye 680RD donkey anti-mouse IgG (1:25,000) (LI-COR, Lincoln, NE, USA) were used as the secondary antibody. Indirect Enzyme Linked Immunosorbent Assay (ELISA) was used to determine rabbit anti-rEtCDPK4 serum titers.

The recombinant His₆-Vp7 protein was expressed from pJS1621 in E. coli BL21 (DE3) by induction with IPTG, and purified by nickel affinity column chromatography using Ni-NTA His•Bind^® Resins (Novagen, Carlsbad, CA, USA). The eluted protein was checked for integrity by 15% SDS-PAGE, and its concentration was determined using Protein Assay kit (Bio-Rad, Hercules, CA, USA). The purified His₆-Vp7 protein was used to raise the anti-Vp7 antibody in a New Zealand rabbit via an intraperitoneal route with 10 μg His₆-Vp7 in sterile 0.85% NaCl formulated with 50% (v/v) Freund complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) in a total volume of 200 μL per dose. After 30 days of primary immunization, the animals were boosted at 10-day intervals for 1–2 times. One week after the last immunization, sera were collected and checked for antibody specificity at a dilution of 1:2000 by Western blot analysis.

For animal immunizations, a solution with 20 mg/ml of BSA (Sigma-Aldrich, St. Louis, MO, USA) was prepared and passed through a 0.2-μm sterilized microfilter. An equal volume of Freund Complete Adjuvant (Sigma-Aldrich, St. Louis, MO, USA) was mixed with the BSA solution and emulsified. The immunization was performed by subcutaneous injections of this emulsion. A total of 0.4 ml/kg was injected on days 0, 14, and 21. On day 28, a total of 0.5 ml of SF was aspirated from carpal joints. Intra-articular injections of BSA (0.5 ml at 20 mg/ml) were bilaterally performed to induce an antigen-mediated immune response. The left carpal joints were used as control (BSA co-administered with PBS) and the right carpal joints were used for exosome-based treatments (BSA co-administered with exosomes). The exosomes were used at the concentration of 500 μg protein/injection in a total volume of 500 μl.

Animal experiments were approved by and performed at the Animal Experiment Center of Yangzhou University (Yangzhou, China). All animal experiments followed the National Institute of Health guidelines for the ethical use of animals in China.
A total of 45, 8-week-old female BALB/c mice (Laboratory Animal Center of Yangzhou University, Jiangsu, China) were divided into three groups, with 15 mice per group. In group 1, mice were SC co-immunized with 50 µg of the FaeG–Fim41a–FanC–FasA MEFA protein combined with 50 µg FedF–FasA–Fim41a–FanC. In group 2, mice were SC immunized with only 100 µg of the FaeG–FedF–FanC–FasA–Fim41a MEFA protein. An equal volume of Freund complete adjuvant (Sigma, USA) was used as the adjuvant with the primary immunization. Each mouse received two booster injections at the same dose as the primary immunization, but with Freund incomplete adjuvant (Sigma, USA). These boosters occurred at 2-week intervals. In group 3, mice were injected only with 200 µL of sterile phosphate buffer saline (PBS) and were used as the control group. All mice were euthanized 2 weeks after the second booster. Blood samples were collected from each mouse both before immunization and at the time of euthanasia.