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 log₂serum 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).

IgG antibody titer (SRBC)

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HAWC