Freund s incomplete adjuvant fia

Two methods were used to produce rabbit polyclonal antibodies against formalin-treated whole-cells. In the first method, formalin-treated B. mallei (fBm) GB18-3 cells were formulated with Ribi TriMix as the adjuvant (Ribi ImmunoChem Research Inc., Hamilton, MT, USA) [13 (link)]. In the second method, formalin-treated B. pseudomallei K96243 (fBpK) cells were formulated with Freund’s complete adjuvant (FCA) or Freund’s incomplete adjuvant (FIA) (Sigma-Alrich, Saint Louis, MO, USA). The general procedure to generate polyclonal antibodies in 2 female NZW rabbits (~2.5 kg) were as follows (Covance Research Products, Denver, PA, USA): prebleed, 21 days before the primary vaccination; primary vaccination, 250 µg of fBpK in FCA; 3 boost (21 days apart) vaccinations starting 21 days after the primary vaccination, 125 µg of fBpK in FIA; terminal bleed, 14 days after the last boost. Antibody (IgG) titers against IRBpK, fBpK, and fBm cells were determined at least twice by ELISA as previously described [14 (link)]. See Table A1 in Appendix A for antibody titers of antibodies used in the present study. No new animals were used at USAMRIID for this report.

N2’-Deacetyl-N2’-(3-mercapto-1-oxopropyl)-maytansine (Mertansine, DM1 compound) was obtained from Abcam (Cambridge, UK). Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). BSA-conjugated DM1 was prepared by Abfrontier (Seoul, Republic of Korea). Protein A and Protein G were purchased from GE Healthcare. T-DM1 (trade name Kadcyla^®) was purchased from Dongwon Pharmaceutical (Daejeon, Republic of Korea) for laboratory research use. Human anti-trastuzumab, a positive control antibody, was purchased from Bio-Rad (Puchheim, Germany). HER2-ECD was obtained from Sino Biological Inc. (Beijing, China). Human anti-Fc-specific antibody conjugated to peroxidase was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Biotin-conjugated Kadcyla and HER2-ECD were prepared by AbFrontier (Seoul, Republic of Korea). Streptavidin-HRP was purchased from BD Pharmingen^TM (San Diego, CA, USA). Carbonate-bicarbonate buffer, TMB substrate, and stop buffer were purchased from Sigma–Aldrich (St. Louis, MO, USA). A 1x PBS-T was purchased from Fluka (Buchs, Switzerland). Individual Crl:CD (SD) rat blank sera were obtained from BioChemed (Winchester, VA, USA).

A healthy 5-year-old female Alashan Bactrian camel was chosen for PEDV immunization. The cell culture medium containing 10⁵ pfu/ml PEDV strain CV777 inactivated by formaldehyde was mixed with equal volume of Freund’s Complete Adjuvant (FCA) (Sigma-Aldrich, USA) and subcutaneously injected into the camel cervical area for the first immunization, and Freund’s Incomplete Adjuvant (FIA) (Sigma-Aldrich, USA) was used for the second and third immunizations. The time interval between immunizations was 10 days, and the camel blood was taken from the jugular vein 10 days after each injection to prepare the serum for evaluation of the PEDV heavy chain antibody (IgG2) titer. The camel was farmed in the isolated Gobi area of the Alxa Left Banner in Inner Mongolia and was provided free access to water and food. The experimental procedures were performed in accordance with the institutional and national guidelines and regulations and were approved by the Animal Care and Use Committee of Inner Mongolia Agriculture University.

Three chickens (White Leghorn, 21 weeks old) were obtained from a commercial breeder (Cuban Centre for the Production of Laboratory Animals, CENPALAB, Havana, Cuba) and were kept individually in cages (128×65×80 cm), water and food were provided (special diet CM 005 Al y Co, CENPALAB, Havana, Cuba) ad libitum, according to general ethical regulations in Cuba, and to a great extent to the recommendation of the 21^st ECVAM workshop on IgY-technology¹¹ .
The immunization of animals was done via left and right breast with 20 uL (approx. 800 ug of C3, 5 ug of C4 and 300 ug of human IgG) from human umbilical cord serum, with frequency of 0, 15, 36, 66 days with volume 1.0 mL of phosphate-buffered saline and PBS was added to umbilical cord serum to complete 500 uL and for mixing with Freund’s Complete Adjuvant (FCA, first immunization, SIGMA, Germany) or Freund’s Incomplete Adjuvant (FIA, booster-immunizations, SIGMA, Germany) in a ratio of v:v (umbilical cord serum-PBS:FCA/FIA).

Hybridoma cells were produced as per the method described by Liu et al. (2014) (link). Briefly, mice were immunized subcutaneously with purified His-EF-Tu protein (50 μg/mouse) suspended in Freund’s complete adjuvant (FCA; Sigma-Aldrich, St. Louis, MI, United States). Following two additional immunizations with the same dose of Freund’s incomplete adjuvant (FIA; Sigma, United States) at 2-week intervals, immunoreactivity against EF-Tu was validated and a final booster immunization of purified His-EF-Tu protein (100 μg/mouse) was given without adjuvant. Four days after booster immunization, mice were euthanized, their spleens removed, and spleen cells isolated using standard techniques. The spleen cells were fused with SP20 myeloma cells to generate hybridomas as described by Hadji-Ghasemi et al. (2003) (link). The immunoglobulin subclass was determined using an IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Sigma-Aldrich) as per the manufacturer’s instructions. Selected cell clones were then cultured in the peritoneal cavities of BALB/c mice to obtain ascetic fluid. McAbs were then purified from the resulting ascites fluid using a Protein G Spin Purification Kit (Pierce Biotechnology, Rockford, IL, United States) and stored at −80°C until further use. The resulting anti-B. melitensis EF-Tu McAb was named BD₆.

The local strain of B. melitensis was grown in 35 mL of Brucella broth (BBL™, UK) in shaking incubator for 4 days at 37 °C. The bacterial concentration in the broth was determined using the standard total plate count method. The cells were re-suspended in sterile PBS to obtain a final concentration of 1 × 10⁹ cfu/mL, were then killed by adding 0.5% formalin and were emulsified with Freund’s complete adjuvant (FCA) (Sigma-Aldrich, US) at 1:1 ratio. One mL of the emulsion was injected subcutaneously into rabbits. Booster doses of the emulsified inoculums, prepared using Freund’s incomplete adjuvant (FIA) (Sigma-Aldrich, US) were injected on days 14 and 21. Finally, the hyperimmune serum was harvested at 28 days post-inoculation. The Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia approved this protocol (AUP No: R019/2014).