Freund s incomplete

Cs16 antiserum was prepared in C57BL/6 mice. Cs16 (50 μg) was formulated with either Freund′s complete (primary) or Freund′s incomplete (two boosts at 2-week intervals) adjuvant (Sigma-Aldrich, USA) and injected subcutaneously into mice. Mice were sacrificed 2 weeks after the final antigen immunization. Immunoglobulins from sera were first precipitated with ammonium sulfate and then purified using Protein A/G PLUS-Agarose Beads (Santa Cruz Biotechnology, USA) according to the manufacturer’s instructions.

DHA and sodium iodide were purchased from J&K Scientific Ltd (Beijing, China). Porcine Tg, LPS, con A, complete Freund’s adjuvant (CFA), and incomplete Freund’s (IFA) adjuvant were provided by Sigma–Aldrich Co. Ltd (St. Louis, MO, USA). PMA was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). IP-10 (CXCL10) was obtained from PeproTech EC Ltd. (Rocky Hill, NJ). CXCR3 plasmid was purchased from GeneCopoeia Inc (Rockville, MD, USA). FLIPR Calcium 6 Assay Kit was provided by Molecular Devices, LLC (Sunnyvale, CA). PI3K antibody, p-PI3K antibody, AKT antibody, p-AKT antibody, NF-kB/p65 antibody, p-NF-kB/p65 antibody, and CXCR3 antibody were purchased from Affinity Bioreagents Co. Ltd (Colorado, USA). Secondary antibodies were purchased from EarthOx (San Francisco, USA).

Six New Zealand rabbits were subcutaneously immunized with 100 µg of each selected peptide in the following order: NT-1 (rabbit #1), RBD3 (rabbit #2), RBD4 (rabbit #3), N (rabbit #4), mixture of all peptides in S protein (rabbit #5) and mixture of all four peptides (rabbit #6) using complete (Life Technologies Cat # P1503) (first immunization) and incomplete Freund’s (Sigma Aldrich Cat # F5506) (second and third immunization) as adjuvants. A total of three immunizations were performed with an interval of ten days each. For immune response evaluation, rabbits were bled before immunizations (preimmune serum) and 15 (first evaluation) and 35 (second evaluation) days after the initial immunization. All collected blood samples were evaluated for immune response evaluation using indirect ELISA.
Immune response evaluation was performed similarly to the abovementioned ELISA with the following specific differences: antigen fixation was performed using 1 µg/mL of each peptide incubated for 2 h at 37 °C, serial dilutions (1:1000–1:16,000) of the sera were performed and incubated overnight at 4 °C. Finally, an anti-rabbit IgG-HRP coupled (Santa Cruz Biotechnology Cat # sc-2357) was used as a secondary antibody and incubated for 2 h at 37 °C.