Anti human igm agarose beads

Serum, diluted 1:3 in PBS, was added to anti-human IgM agarose beads (Sigma, A9935) for 1.5 h at RT. Two depletion rounds were conducted, using a bead volume that was twice the volume of the serum. Beads were pre-washed with PBS and supernatants were removed following a centrifugation (5 min 400 g). Complete IgM removal was verified by YF17D IgM and IgG ELISA.

Streptavidin-coated paramagnetic beads (100 μl, 1 μm diameter; Life Technologies) were saturated with 20 μg of enzymatically monobiotinylated DBLMSP or a Cd4 tag-alone control, isolated with a magnet, and washed three times with PBS before incubating with 1 ml of filtered human serum (Sigma) for one h at 4 °C. Beads were washed four times with 1 ml of PBS and eluted with 200 μl of 1% SDS. 20 μl were resolved by SDS-PAGE under reducing conditions and stained with SYPRO Orange (Sigma), and the gel image captured on a Typhoon 9400 phosphorimaging device (GE Healthcare).
Anti-human IgM agarose beads (Sigma) were incubated with long term parasite culture supernatants from the IT4 var1 and var13 strains grown in the presence of human serum or control culture medium without parasite for 1 week at 37 °C. After five washes in PBS, the beads were resuspended in loading buffer in the presence or absence of DTT, and eluates were blotted onto nitrocellulose membranes (Amersham Biosciences Protran) followed by blocking in PBS, 0.1% Tween 20, 5% nonfat milk powder) and incubated for 1 h with a rabbit anti-full-length DBLMSP antibody at a 1:100 dilution. After further washes, the membrane was incubated with an anti-rabbit HRP-conjugated IgG secondary antibody (1:1000; Sigma) and developed using 3′,3′-diaminobenzidine (DAKO) according to the manufacturer's instructions.