96 well luminex multi screen bv 1.2 micron assay plates

Luminex beads were purchased from Luminex Corporation (Austin, TX) and 96-well Luminex Multi-screen BV 1.2 micron assay plates were acquired from Millipore Corporation (Billerica, MA). NANP peptide (0.5 μg) or a recombinant protein representing the C-terminal (C-term) region of CSP (0.4 μg) were coupled to 5x10⁶ beads according to the manufacturer’s instructions. Representative serum samples (high and low responders) were tested at a series of dilutions. A titration curve was drawn and a linear range dilution was chosen. All serum samples were run at this one selected dilution. Typically sera were diluted 1/1000 in PBS containing 1% BSA (assay buffer). Fifty μl of the sample was added to the wells along with 50 μl of assay buffer containing 3,000 beads coated with NANP and 3000 C-term protein coated beads (with two different bead signatures). The plates were agitated on a shaker for 1 h at RT and washed in assay buffer containing 0.05% Tween-20. 100 μl of assay buffer containing PE-labeled-(mouse IgG subclass)-specific antibody (Jackson Immunoresearch; West Grove, PA) was then added to the wells and the plate was agitated for an additional hour. The plate was washed and median fluorescence intensity (MFI) was measured for ~100 beads per well using the Luminex 200 system (Luminex Corporation, Austin, TX).

CSP specific monoclonal antibodies used to characterize recombinant CSP were generated in mice against a full-length CSP recombinant protein [36] (link) and showed positive reactivity to Pf sporozoites by IFA and western blot (Dr. Ted B. Hall, personal communication). PE-anti-mouse IgG, PE-anti-mouse IgG1, PE-anti-mouse IgG2c and PE-anti-mouse IgG2a were obtained from Jackson Immunoresearch (West Grove, PA). Luminex beads and Luminex Streptavidin beads were purchased from Luminex Corporation (Austin, TX) and 96-well Luminex Multi-screen BV 1.2 micron assay plates were acquired from Millipore Corporation (Billerica, MA). Antigens were coupled to the Luminex beads according to the manufacturer’s instructions. PerCP anti-CD3 (clone 145-2C11), Pacific Blue anti-CD4 (clone RM4-5), Horizon V500 anti-CD8 (clone 53-6.7), Alexa 700 anti-CD44 (clone IM7), FITC anti-IL-2 (clone JES6-5H4), APC anti-IFN-γ (clone XMG1.2), anti-CD16/CD32 FcR Block (clone 2.4G2), anti-CD28 (clone 37.51), anti-CD49d [clone 9C10(MFR4.B)], Cytofix/Cytoperm and Golgi Plug were all obtained from BD Biosciences (San Jose, CA). LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation was purchased from Invitrogen (Camarillo, CA). Phorbol 12-myristate 13 acetate (PMA) and Ionomycin were obtained from Sigma Chemicals (St. Louis, MO).