Freund s adjuvant

To generate polyclonal antibodies against rTgEF-1α, SD rats were immunized subcutaneously with either 200 μg of recombinant TgEF-1α protein or PBS (control) emulsified with an equal volume of Freund’s complete adjuvant (Sigma–Aldrich, UK). Two weeks later, booster immunizations were done using either 200 μg of recombinant TgEF-1α protein or PBS emulsified with incomplete Freund’s adjuvant (Sigma–Aldrich, UK). Second booster immunizations were done 2 weeks later. Ten days after the second booster immunizations, rats were sacrificed and blood collected by cardiac puncture. Sera were extracted and affinity purified to monospecificity by affinity column chromatography as previously described (Witola et al., 2006 (link)).

Anti-BoTRAP2-1 sera were raised in New Zealand white rabbits by five immunizations with a moderate amount of recombinant protein and Freund’s adjuvant (Sigma, Shanghai, China). The sera were collected when the serum titer reached an appropriate value. Total immunoglobulin Gs (IgGs) were purified from the collected sera by using a Protein A chromatography column (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions and then stored at −20 °C.

Rabbits were immunized with 2 ml (0.5 mg/ml) of PcrV_NH formulated with 2 ml Freund's adjuvant (Sigma) at 0, 14, and 21 days. Seven days after the final immunization, antibodies in the sera were collected and purified. To evaluate protective efficacy of PcrV_NH-specific antibodies in vivo, 30 mice were equally divided into three groups. Then, each group was intraperitoneally injected with 1.0 mg of anti-PcrV_NH antibodies, non-specific rabbit IgGs or PBS. Four hours later, all mice were challenged with a lethal dose of PA XN-1, and survival was observed every 12 h for 7 days.

Four subcutaneous immunizations with 90 μg of a protein representing the region 872–1296 of the Botulinum A₁ Heavy Chain were performed in a male cynomolgus macaque (Macaca fascicularis) in Freund’s adjuvant (Sigma Aldrich). This recombinant BoNT/A₁-HC fragment, corresponding to the binding domain of BoNT/A₁, was prepared as previously described [42 (link)]. The three first immunizations were administered at one month intervals and the fourth immunization was administered four months after the third.

A polyclonal antibody was raised against Mhp Eno by subcutaneously immunizing 1-month-old New Zealand white rabbits. Each rabbit was immunized three times with 1 mg of Mhp Eno emulsified in Freund's adjuvant (Sigma, USA) at 2-week intervals. Sera were collected 1 week after the third immunization.

Specific-pathogen free female BALB/c mice (6 weeks old) were obtained from the Laboratory Animals Centre, National University of Singapore. Mice (10 per group) were vaccinated subcutaneously at days 0 and 28 with 100 µL (HA titer, 128) of inactivated RG-H5N3, RG-H5N3HA_M1, RG-H5N3HA_M2 viruses with Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Phosphate-buffered saline (PBS) was administered with the same adjuvant as a reference vaccine control. The mice serum collected 21 days after dose 2 (day 49) was evaluated by hemagglutination inhibition (HAI) titer [17 (link)], anti-H5HA (A/Indonesia/5/2005) specific antibody titers by indirect ELISA [13 (link)] and VMN [16 (link)] against influenza viruses. In a separate study, mice (6 per group) were vaccinated with inactive RG-H5N3 or RG-H5N3HA_M1 or RG-H5N3HA_M2 vaccine with Freund’s adjuvant on days 0 and 28. Four weeks after the final immunization, mice were anesthetized intraperitoneal (i.p.) with ketamine (100 mg/kg)/xylazine (20 mg/kg) and intranasally challenged with 50 μL (25 μL per naris) of 5 50% mouse lethal dose (MLD₅₀) of A/Hubei/1/2010 RG-H5N1 virus (clade 2.3.2.1). Mice were observed daily to monitor body weight, clinical signs of disease and mortality until day 14 after challenge. Mice were humanely euthanized with CO₂ inhalation if their body weight dropped to 75% of baseline weights.