P nitrophenyl phosphate substrate

The HIS-tagged TGF-β1 (HIS.TGF-β1) ligand was constructed by amplifying the TGF-β1 cDNA by PCR with the primer sequences AAG CTT GCT ATC CAC CTG CAA GAC TAT and CTC GAG CGC TGC ACT TGC AGG AGC GCA. The amplified fragment was then cloned into the HindIII and XhoI restriction sites of pSecTag2 vector (Invitrogen). HIS.TGF-β1 protein was produced by transient transfection into HEK-293 T cells. After 2 days, conditioned medium was collected and added to control or FUT8-KO MDA-MB-231 cells endogenously expressing TGF-β receptors for 4 h at 4 °C. After stringent washing, bound HIS.TGF-β1 protein was detected with AP (alkaline phosphatase)-conjugated anti-HIS antibody. After washing, cells were lysed for 10 min on ice in lysis buffer (25 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM EDTA, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 10% glycerol, and 1% Triton X-100). Cell lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C. The bound AP protein was reacted with p-nitrophenyl phosphate substrate (Sigma) to quantify AP binding in cell extracts.

Plasma samples were assayed using the CHI3L1 Duoset ELISA kit (R&D Systems) according to the manufacturer’s instructions with the following changes: assays were performed in 50 μL/well; plasma samples were incubated overnight at 4°C; and ELISAs were developed using Extravidin®-Alkaline Phosphatase (Sigma, 1:1000, 45 min) followed by addition of p-Nitrophenyl phosphate substrate (Sigma) and optical density readings at 405 nm.

Maxisorp plates (Nunc-Immuno) were coated at 4 °C overnight in bicarbonate buffer with the anti-PfRH5 mouse mAb 2AC723 (link) at 5 μg/mL. Plates were washed with PBS/0.05% Tween-20 (PBS/T), blocked in Casein Blocker in PBS (Thermo Scientific) for 1 h at room temperature (RT), and washed again prior to addition of diluted PfRh5 protein samples in duplicate. Serially diluted pure PfRH5 v1.0 in the range 200 ng/mL to 1.56 ng/mL was added to each plate to generate a standard curve. Plates were incubated at RT for 2 h and then washed prior to addition of rabbit serum from animals immunized with PfRH5 (3D7) viral vectored vaccine7 (link) diluted at 1:1000 in Casein Blocker. After a further 1 h incubation and subsequent wash, alkaline phosphatase-labelled goat anti-rabbit IgG (whole molecule) (Sigma) was added to each well at a 1:5000 dilution in Casein Blocker and plates were left to incubate for a final hour. Bound antibodies were detected by addition of p-nitrophenyl phosphate substrate (Sigma) diluted in diethanolamine buffer (Thermo Scientific). The optical density at 405 nm (OD₄₀₅) was read using an ELx800 microplate reader (BioTek, UK) and a four parameter fit was generated from standard curve values with Gen5 ELISA software v1.10 (BioTek, UK). This curve was used to convert the absorbance of diluted PfRH5 samples into concentration of PfRH5 protein.

On days 7 and 14, cellular alkaline phosphatase (ALP) activity was measured. Cell medium was removed, cells were washed with alkaline buffer solution (5 M NaCl, 1 M Tris-Cl pH 9.5, 1 M MgCl₂), then 250 μL of 0.1% Triton X-100 in alkaline buffer was added. Plates were stored at −80 °C and subjected to three freeze–thaw cycles to fully lyse all cells. Once defrosted, 50 μL of each lysate was transferred, in duplicate, into a 96-well assay plate and supplemented with 200 μL of test solution made of alkaline buffer solution and p-nitrophenyl phosphate substrate (both from Sigma-Aldrich, U.K.). Standards were prepared from p-nitrophenol diluted with alkaline buffer solution. Each plate was then covered in foil (to protect from light) and incubated for 30 min at 37 °C. A total of 50 μL of stop solution (3 M NaOH) was added to the wells to terminate the reaction and absorbance was then read at 405 nm using a Tecan GENios microplate reader A-5082, (Tecan, Grodig, Austria). Finally, a PicoGreen assay (Thermo Fisher Scientific, UK) was conducted on the same cell lysates according to manufacturer’s protocol to measure the amount of dsDNA. This allowed ALP activity to be normalised to cell number.