Endpoint name in study: SRBC-specific IgG antibody titer - Animals (16/dose) were immunized on the 11th day of dosing by intravenous injection of 4.0 × 107 sheep red blood cells (SRBCs) in 0.2 mL sterile saline. Five days later, 8 animals/dose were anesthetized with carbon dioxide and exsanguinated by neck vein transection. Blood was collected and held at room temperature for 30 min, centrifuged at 4°C to separate serum, and serum was frozen at –80°C until analysis of SRBC-specific IgM. Two weeks after primary immunization, the remaining 8 animals/dose were given a booster immunization of SRBCs (4.0 × 107). Five days later, animals were anesthetized with carbon dioxide and exsanguinated by neck vein transection. Blood was processed as described above for later analysis of SRBC-specific IgG. The relative serum titers of SRBC-specific IgM and IgG antibodies were measured by ELISA as described below.
IgM titers were determined as described previously (DeWitt et al. 2005). Briefly, flatbottom 96-well Immunolon-2 ELISA microtiter plates (Dynatech Labs, Chantilly, VA) were coated with 125 μL of 2 μg/mL of SRBC membrane [1.46 mg/mL stock solution diluted in phosphate-buffered saline (PBS); prepared according to Temple et al. (1995)] and then incubated at 4°C for at least 16 hr. Each plate included 20 wells that were coated with pooled serum collected from healthy mice 5 days after primary immunization with SRBCs, and 16 wells contained 100 μL PBS as blanks. After washing, blocking of nonspecific binding, and addition of serum samples (serially diluted from 1:8 to 1:4,096), secondary antibody (goat anti-mouse IgM horseradish peroxidase; Accurate Chemical and Scientific Corp., Westbury, NY) was added. Following three washes and addition of substrate [one tablet of 2,2´-azino-di-(3 ethylbenzthiazoline sulfonic acid) (ABTS; Sigma Chemical Company, St. Louis, MO); added to 50 mL phosphate-citrate buffer with one tablet of urea hydroxide peroxide (Sigma) in 100 mL of distilled water], plates were incubated for 45 min at room temperature and then read at 410 nm on a SpectraMax 350 plate reader (Molecular Devices, Sunnyvale, CA).
The concentration of SRBC-specific IgG was evaluated using the same quantitative assay as for IgM with the following exceptions: The internal control for IgG was generated by serial dilution (from 1:8 to 1:4,096) of 100 μL pooled serum collected from healthy mice 5 days after a second immunization with SRBCs; F(ab´)2 goat anti-mouse IgG horseradish peroxidase (Accurate Chemical and Scientific Corp., Westbury, NY) was used as the secondary antibody. Absorbance was read on a SpectraMax 350-plate reader at 410 nm. Both IgM and IgG antibody titers were processed using SOFTmax Pro software (Molecular Devices) to determine the log2 serum titer for an optical density of 0.5 units from the log–log curve of optical density versus dilution, as described by Temple et al. (1995). |