Collect PBS and dislodged cells by suction at the edge of the dish. 4 Repeat the washing process RNF55 three times. 3.5.2 Automated Washing Station An automated washing station was designed to allow consistent plate washing to reduce the variation between plates. acquired by multigravid women. Currently, an international effort to develop a vaccine to prevent pregnancy malaria focuses on a member of the PfEMP1 variant surface antigen family named VAR2CSA. VAR2CSA is preferentially expressed by placental parasites and parasite isolates selected for binding to CSA [4], and women acquire antibodies to VAR2CSA over successive pregnancies as they develop resistance to placental malaria. VAR2CSA is a large protein of about 350 kDa composed of six extracellular Duffy binding-like (DBL) domains, and is too large to manufacture as an intact molecule. Therefore immunogens are being considered that incorporate one or a combination of VAR2CSAs DBL domains, with or without adjacent interdomain regions. One of the major challenges in developing a pregnancy malaria vaccine (PMV) is identifying a domain or domain combination that can elicit broadly reactive antibodies. In endemic areas women acquire strain-transcending antibodies that block parasite adhesion to CSA and this property has been an important criterion in selecting candidates. A number of studies have evaluated PMV candidates by assessing the level of inhibition of adhesion by antibodies raised to recombinant forms of VAR2CSA domains or full-length protein. Several platforms for binding inhibition assays have been described, including a high-throughput assay in 96-well plates of tritium-labeled parasites [5] and a flow-based assay [6]. Here, we focus on the static Petri dish-based assay [7]. This assay format can be adapted to any lab that is minimally equipped for short-term parasite culture, a centrifuge, and a microscope. Therefore, the assay can be used MBM-55 and has been used to analyze anti-adhesion activity on fresh parasite isolates that can thus support the selection of the optimal PMV candidate. 2 Materials 2.1 Reagents Trophozoite-stage parasites at 5C20 % parasitemia, 0.5 % hematocrit (Subheading 3.1). 100 mm 20 mm polystyrene Petri dishes (Falcon MBM-55 351029) (Subheading 3.3). 2.2 Equipment Tabletop centrifuge (that allows for centrifugation at 1800 CS2 available from MR4 (MRA-96). 2 Fresh isolates obtained from the peripheral blood of pregnant women (for 5 min and resuspend with RPMI 1640 to 50 % hematocrit. 6 Add two volumes of gelatin solution and incubate for 30 min in a 37 C water bath. In the absence of MBM-55 a water bath, an incubator set at 37 C can be used. 7 Mature parasites remain in the upper layer while uninfected erythrocytes and ring-stage parasites form rouleaux and descend to the bottom layer. 8 Transfer the upper layer to a new tube, pellet the blood containing mature parasites, and wash three times with RPMI 1640 media. 9 Prepare and read a thin blood smear using methanol fixation followed by Giemsa staining: if parasitemia is 20 %, dilute to 20 % with uninfected red blood cells. 3.2 Plate Preparation Using a template, draw circles on the back of a polystyrene Petri dish and label the wells appropriately. To prepare a template, use a circular Petri dish-sized piece of material (e.g., MBM-55 plastic, cardboard, paper) that contains twenty 10 mm circles drawn in 18 increments around the perimeter. Each circle will be able to accommodate approximately 20 l of liquid ((all spins are performed for the same time and force). Wash column/beads using 400 l binding buffer; spin and discard flow through. Repeat one more time. Apply the centrifuge column cork to the bottom of the column. Add 100C450 l of plasma/serum sample on top of the beads (optimal volume is 150 l); cap the column (Parafilm? wrap is recommended to prevent leakage). Incubate with gentle rotation end over end, for 20C30 min at 4 C. Remove cork from column and spin; discard flow through (this can be saved to MBM-55 determine binding efficiency). Wash using 400 l of binding buffer, inverting the column several times after buffer application. Discard flow through after spin..