Binding of DBL domains of 3D7 var2CSA to CSA and recognition by mouse sera raised against DBL3γ of FCR3 var1CSA

ConstructFrequency of reactivity (%)aBinding efficiency (%)b
DL6Anti-rDBL3γ antibodiesCSACSBCSC
pRE4-DBL3γ74 ± 463 ± 472 ± 500
pRE4-DBL1X70 ± 80000
pRE4-DBL2X60 ± 6058 ± 600
pRE4-DBL3X66 ± 857 ± 360 ± 1000
pRE4-DBL4ε58 ± 40000
pRE4-DBL5ε63 ± 70000
pRE4-DBL6ε54 ± 60000
pRE483 ± 40000
  • a The DBL3γ domain of FCR3 var1CSA and all the DBL domains (DBL1X to DBL6ε) of 3D7 var2CSA were expressed on the surfaces of mammalian 293T cells as fusions to HSV gD by use of the transfection vector pRE4. Mouse monoclonal antibody DL6 directed against HSV gD sequences in the fusion proteins and mouse polyclonal sera raised against DBL3γ of FCR3 var1CSA were used to detect expression of DBL domains on 293T cell surfaces. Preimmune mouse serum does not react with any cells transfected with any of the DBL constructs tested. Around 1,000 to 1,500 293T cells were scored for recognition by DL6 and anti-rDBL3γ mouse sera in immunofluorescence assays, and the frequencies of reactivity were determined for both antibodies. Results represent the average and standard deviation of three independent experiments.

  • b Transfected 293T cells were incubated with Bio-CSA, Bio-CSB, and Bio-CSC to allow binding followed by incubation with anti-biotin sera raised in mice. Anti-mouse IgG chicken IgY conjugated with Alexafluor 488 (Molecular Probes) was used to detect binding of biotinylated CSA, CSB, and CSC. Around 1,000 to 1500 293T cells stained with DAPI were scored for staining with Alexafluor 488. The binding efficiency was calculated as follows: binding efficiency (%) = [(number of Alexafluor 488-stained 293T cells)/(number of DAPI-stained 293T cells] × 100. Where the binding efficiency is reported as zero, no Alexafluor 488-stained cells were seen in the entire well. Results reported are average ± standard deviation from three independent experiments.