Antibody-dependent cell inhibition (ADCI assay)

The antibody-dependent cell inhibition (ADCI) assay was performed with purified IgGs from AMSP-Fu₃₅ rabbit sera according to procedure described earlier [37]. For this assay, THP1 monocyte culture stocks were obtained from American Type Culture Collection (ATCC) and maintained in complete RPMI medium containing 10% Fetal Bovine Serum (HIMEDIA). Briefly, tightly synchronized P. falciparum 3D7 schizont-stage parasites (0.5% parasitemia and 2% hematocrit) were co-cultured in a 96-well flat-bottom micro-culture plate with human monocytes (2×10⁶ monocytes per ml) in complete medium (RPMI medium containing 0.5% albumax). Purified antibodies at a concentration of 50μg/ml were added to these cultures and incubated at 37°C for 96hr in mixed-gas environment. After 48 and 72hrs of growth, 50μL of complete medium was added to each well. After 96hrs of growth, parasitemia was estimated by a two colour staining with ethidium bromide and anti-HLA-ABC antibodies and acquiring the stained cells using a FACS Calibur cytometer (BD Bioscience). Control wells consisted of (i) parasite alone, (ii) parasite and control IgG (purified from naïve rabbit sera), (iii) parasites and monocytes, (iv) parasite and purified IgG without monocytes, and (v) parasites, control IgG, and monocytes. The specific growth inhibition index (SGI) was calculated as follows: 1− [(percent parasitemia with monocytes and test antibodies/percent parasitemia with test antibodies)/ (percent parasitemia with monocytes and control IgG/percent parasitemia with control IgG)] × 100.

For reversal assay, total IgGs from AMSP-Fu₃₅ were pre-incubated with three different concentrations (25μg/ml, 50μg/ml, 100μg/ml) of recombinant AMSP-Fu₃₅, PfMSP-1₁₉, PfMSP-3₁₁ and PfAARP for 1hr at 37°C following which the assay was performed as described above.