Incomplete adjuvant

After mixing the full-length SFTSV N protein (1 mg/mL) with an equal volume of incomplete adjuvant (Sigma, USA), 150 µL of the mixture was injected into each footpad of BALB/c mouse three times at three-week intervals. After immunization, the lymph nodes were collected to obtain B cells that then were fused with Sp2/O myeloma cells to produce hybridoma cells [30 (link)]. The hybridoma cells were incubated for two weeks in a 96-well plate. Cell culture medium containing the antibodies was transferred into a new 96-well plate coated with 1 µg/mL of SFTSV N protein to test their immune responses using an indirect ELISA method. The cells confirmed to produce SFTSV N protein-specific antibodies were processed by serial dilution, transferred to a new 96-well plate, and incubated for one week. The ELISA test was performed again to confirm antibody production by the hybridoma cells. The verified cells were then cultured up to a 250 mL volume through sequential scale-up and were concentrated as appropriate. About 0.5 mL of the antibody-producing hybridoma cells (1 × 10⁶) were injected into a BALB/c mouse intraperitoneally, and monoclonal antibodies were harvested and purified from ascites fluid.

Oxycodone was obtained through the NIDA Drug Supply Program and Sigma (St. Louis, MO). Oxycodone doses and concentrations are expressed as the weight of the base. Alum (Alhydrogel85, Brenntag Biosector, (Frederikssund, Denmark), Freund’s complete (Calbiochem, San Diego, CA) and incomplete adjuvant (Sigma-Aldrich, St. Louis, MO), and monophosphoryl lipid A (MPLA SM VacciGrade, InvivoGen, San Diego, CA) were used according to manufacturer instructions.

5 Balb/c mice (8–12 weeks of age) per immunogen were immunized by intraperitoneal or foot pad injections using either sGP or GP proteins (total of 20 mice: 5 mice x 2 injection sites x 2 antigens) emulsified in Freund's complete adjuvant (Sigma) (first injection) or incomplete adjuvant (Sigma) (boost injections) (5–10 μg of proteins every 2 weeks). Three days after the last injection, spleen cells (intraperitoneal injections) or popliteal lymph node cells (foot pad injections) were fused with SP2/0 myeloma cells (Sp2/0-Ag14 (ATCC CRL-1581), using polyethylene glycol (PEG 1500) (Sigma). Hybridomas were grown in DMEM culture medium (Sigma) containing 20% Fetal Calf Serum (FCS) (Eurobio, France), penicillin (100 IU/ml) and streptomycin (100 μg/ml) and supplemented with hypoxantine (1×10⁻⁴ M), aminopterin (4×10⁻⁷ M) and thymidine (1.6×10⁻⁵ M) (HAT) (Sigma). After ten to fourteen days of culture, secreting hybrids were identified by analysis of culture supernatants by indirect enzyme-linked immunosorbent assay (ELISA). Hybridomas from selected antibody were cloned by limiting dilution and processed according to conventional methods. Clones secreting antibody of desired reactivity were expanded in 25 and 75 cm² flasks (Nunc, Denmark), harvested and cryopreserved in 40% FCS, 10% Dimethylsulfoxide (DMSO) (Sigma) and 50% RPMI-1640 culture medium (Sigma).

To prepare mouse anti-GST sera, 6 mice for each GST were used and each mouse was injected intraperitoneally with 0.5 ml of 200 μg/ml of recombinant GST completely mixed with an equal volume of Freund’s Complete Adjuvant (Sigma-Aldrich) to give each mouse 100 μg recombinant protein. Immunization was repeated 14 and 28 days after the first immunization; however, recombinant GST was mixed with incomplete adjuvant (Sigma-Aldrich). All sera were collected 14 days after the last immunization. Antisera were tested using Western blotting, using both recombinant GSTs and tick protein.

HA or HB mice were immunized with 200 μL of recombinant human FVIII (rhFVIII, Baxter, Deerfield, IL, USA; 500 U/kg) or recombinant human FVIII (rhFIX, Pfizer, New York, NY, USA; 200 U/kg) in the presence of complete adjuvant (Sigma) through intraperitoneal injection to induce anti‐hFVIII or anti‐hFIX antibodies, respectively. Another two injections were performed similarly to the first except using an incomplete adjuvant (Sigma) at 3‐week intervals. hFVIII and hFIX inhibitors were determined using a modified Bethesda assay.²¹ Briefly, sequential dilutions of mouse plasma were incubated with equal volumes of 1 U/mL rhFVIII or rhFIX at 37°C for 2 h. Residual FVIII:C and factor IX activity were measured through FVIII and FIX chromogenic assays (Hyphen BioMed, Neuville‐sur‐Oise, France), respectively.