Mouse anti mbp mab

ELISA plates (96-well) were coated with 10 µg/mL of S. exigua BBMVs (SeBBMVs) in carbonate buffer at 4 °C overnight and blocked with 3% BSA for 2 h. Increasing amounts of purified DpCPV virions (0.01–100 µg) or MBP-fusion proteins (0.02–4 µM) in a total volume of 100 µL were added to the wells and incubated for 1 h. The wells were washed with PBST (4 mM KH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl, 0.05% Tween 20, pH 7.4) and incubated with a rabbit anti-DpCPV virion antibody or a mouse anti-MBP mAb (Sigma-Aldrich, St. Louis, MO, USA) followed by horseradish peroxidase (HRP) labeled anti-rabbit IgG (Sigma-Aldrich) or HRP labeled anti-mouse IgG (Proteintech, Wuhan, China). The reactions were visualized using TMB reagent (Beyotime, Nantong, China), stopped by addition of 2 M H₂SO₄, and the absorbance was measured at 450 nm using an ELx808 absorbance reader (BioTek, Winooski, VT, USA). The non-specific binding of BSA coated wells, instead of BBMVs, was subtracted from the total binding value. For the competition assays, the concentrations of the MBP-fusion proteins were kept stable (0.3 µM), while the DpCPV virions were serially diluted (0–10 µg). The protein-virion mixtures were added to the wells; after absorption for 1 h, the bound MBP-fusion proteins were detected as described above.

B. mori BBMVs (BmBBMVs) (10 μg/lane) were separated by 10% sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and blocked in 5% BSA in phosphate buffered saline (PBS) for 2 h. The membranes were incubated with 5 μg/mL of purified MBP-MTase, MBP-VP4, or MBP proteins (as the negative control) for 1 h at room temperature. After washing with PBST buffer, the membranes were incubated with a mouse anti-MBP mAb (Sigma-Aldrich) followed by HRP-labeled anti-mouse IgG (Proteintech), and visualized using ECL reagent (Beyotime).

Protein G Dynabeads (Thermo Scientific, Waltham, MA, USA) (20 μL) were immobilized with 5 μL of mouse anti-MBP mAb (Sigma-Aldrich) overnight at 4 °C, mixed with 20 μg of MBP or MBP-MTase with 20 μg of ALP or BSA in a total volume of 500 μL PBS and then incubated at 4 °C for 1 h. The beads were washed five times with 500 μL of PBS with a magnet, suspended in 30 μL of 2 × sodium dodecyl (lauryl) sulfate (SDS) sample buffer, boiled for 5 min, and loaded on a 10% SDS-PAGE gel for Western blotting analysis using a mixture of mouse anti-MBP (Sigma-Aldrich) and mouse anti-His (Proteintech) mAbs. Prokaryotically expressed BmALP protein was used as the control.