Irdye goat anti human

rPv32 was separated using 12% SDS-PAGE after denaturation with the reducing agent β-mercaptoethanol in sample buffer and then stained with Coomassie brilliant blue. For western blot analysis, recombinant proteins were transferred electrophoretically to PVDF membranes (Millipore Corp., Bedford, MA, USA) and incubated with blocking buffer (5% non-fat milk in PBS containing 0.2% Tween 20 and PBS/T) for 1 h at 37 °C. After blocking, either anti-GST antibody, mouse and rabbit immune sera or mixed patient sera was diluted with PBS/T 200 times. Secondary IRDye^® goat anti-mouse (1:10,000 dilution), IRDye^® goat anti-rabbit (1:20,000 dilution) or IRDye^® goat anti-human (1:20,000) (LI-COR^® Bioscience, Lincoln, NE, USA) were used to detect GST-tagged recombinant protein and immune serum of a specific quality. Sera from healthy people from the ROK and PBS-immunized rabbit serum were used as controls. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and analyzed by Odyssey software (LI-COR, Inc.).

A total of 10 μg of each recombinant PvGAMA protein was prepared in reducing sample buffer, separated by 12% SDS-PAGE, and stained with Coomassie brilliant blue. P. vivax parasites rich in schizonts were harvested from 10 ml of a patient blood sample (parasitaemia >0.1%) using the Percoll-gradient method, and the parasite proteins were extracted in SDS-PAGE loading buffer. One-tenth of the parasite lysate was loaded in each lane and separated by 12% SDS-PAGE. For western blot analysis, the proteins were transferred electrophoretically to PVDF membranes (Millipore Corp., Bedford, MA, USA) and incubated with blocking buffer (5% nonfat milk in PBS containing 0.2% Tween 20, PBS/T) for 1 h at 37 °C. The blots containing recombinant proteins were then incubated for 1 h at 37 °C with either anti-Penta-His antibody (QIAGEN, Hilden, Germany), anti-PvGAMA-Ecto, anti-PvGAMA-Tr1, pooled vivax infected patient sera, pre-immunized rabbit sera, or pooled non-infected human sera diluted 1:1000 in PBS/T. The membranes were washed with PBS/T and incubated with IRDye^® goat anti-rabbit or IRDye^® goat anti-human (LI-COR Bioscience, Lincoln, NE, USA) to detect recombinant proteins according to the manufacturer's instructions. The data were scanned using an Odyssey infrared imaging system (LI-COR Biosciences) and analysed with Odyssey software (LI-COR Bioscience).