Heparan sulfate from bovine kidney

Heparan sulfate-binding assay was performed as described before [54] with some modifications. Briefly, Maxisorp flatbottom 96-well microplates (Jet Biofil, Guangzhou, China) were coated with 100 µl/well of 10 µg/ml (diluted in water) of heparan sulfate from bovine kidney (Sigma-Aldrich Co.), and the control wells were incubated with 100 µl of 10 µg BSA/ml, and all plates were incubated at 37 °C overnight. Next, the plates were washed with 0.05% Tween-20 (Sigma-Aldrich Co.) in PBS (PBS-T) and then were blocked with a 1% bovine serum albumin (BSA) in PBS at room temperature (RT) for an hour. After washing steps, the plates were incubated with the different concentrations of rPvTRAP antigen (starting at 60-0.625 µg/ml) in PBS at RT for 2 h. After washing with PBS-T, the binding of rPvTRAP to heparan sulfate was detected by the addition of 100 µl mouse polyclonal antibodies against rPvTRAP (obtained 38 days after the first immunization) for 2 h, followed by goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:25,000; Sigma-Aldrich Co.) at RT for an hour. The reaction was developed using o-phenylenediamine/H 2 O 2 (OPD, Sigma-Aldrich Co.), as a substrate, and stopped with 2N H 2 SO 4 . The optical density (OD) at 490 nm was read using a microplate reader (BioTek, Winooski, VT, USA).