MagPix multiplex bead array assay
malaria
MALARIA, SECONDARY
IgG sub-types 1–4 against 15 Using a multichannel pipette, 50μl of prepared plasma at a dilution of 1in1000 in buffer B and pooled hyper immune control (27 adults from malaria endemic areas) was added to the beads; two wells had PBS added in them as blanks. The plates were then put on a shaker at 600 rpm for 90 minutes after covering the plates with aluminum foils at room temperature. After incubation, the plates were placed on a magnetic separator for 2 minutes, supernatant poured off by a rapid inversion with a sharp shake followed by a gentle blot on a paper towel. The plates were then washed three times using 200ul wash buffer. 50μl of a secondary antibody specific for an IgG sub-type (IgG1, IgG2, IgG3 and IgG4) with concentration of 1in1000 for IgG1,IgG2 and IgG4 while secondary antibody against IgG3 was diluted at 1in 2000 in dilution buffer and added to each well containing bound primary antibody-antigen complex, blank wells, positive control wells and negative control wells. Incubation was done for 60 minutes in a shaker. The plate was put on a magnetic separator for 2 minutes, supernatant poured off and washed three times using wash buffer to remove unbound antibodies.Using a multichannel pipette, 50μl of 1in 200 R-Phycoerythrin-conjugate AffinPure F (ab’) goat anti-human IgG (