Microtainer sst blood collection tubes

Six- to 8-week-old female BALB/c mice were immunized by intramuscular injection (i.m.) twice, 2 weeks apart, with buffer (50 µL of 10 mM Tris–HCl, pH 8.0) or PapMV-SrtA-M2e, PapMV-sM2e, PapMV-SrtA-T20 or PapMV-SrtA with free T20 peptide. Blood samples were collected before each immunization on days 13 and 27. Serum was separated from the blood by centrifugation in BD Microtainer SST blood collection tubes (BD, East Rutherford, New Jersey, USA) for 2 min at 10,000×g. Total IgG and IgG2a serotype endpoint titers against M2e and T20 peptides in the sera of immunized mice were determined by enzyme linked immunosorbent assay (ELISA) as described below.

Six to 10 week old female BALB/c mice were immunized by intramuscular injection (i.m.) twice, 3 weeks apart. Each group contained five mice. Group 1 received control buffer (50 μL of 10 mM Tris–HCl, 150 mM NaCl pH 8.0); Group 2 received PapMV-C and free M2e peptide; Group 3 received PapMV-C coupled to M2e; Group 4 received PapMV-N and free M2e peptide; Group 5 received PapMV-N coupled to M2e. To assess the humoral response to the M2e peptide, blood samples were collected before each boost-immunization on days 20 and 42. Serum was separated from the blood by centrifugation in BD Microtainer SST blood collection tubes (BD, East Rutherford, NJ, USA) for 2 min at 10,000× g. The immunoglobulin G (IgG) 2a endpoint titer to M2e peptides in the sera of immunized mice was determined by enzyme linked immunosorbent assay (ELISA) as described previously [33 (link)]. Briefly, 96-well flat-bottom nunc^TM MaxiSorp plates (VWR, Radnor, PA, USA) were coated overnight at 4 °C with the M2e peptide at 1 μg/mL. Two-fold step serial dilutions of mice sera, starting at 1 in 50, were prepared. Primary antibodies were revealed using peroxidase-conjugated goat anti-mouse IgG2a (Jackson Immunoresearch, West Grove, PA, USA). Results are expressed as antibody endpoint titers greater than threefold OD_450nm of the background value consisting of a pool of pre-immune sera.