Ap conjugated anti rabbit igg

Chimeric MAb and rabbit sera were tested for specificity by Western blot. Lysates containing equal concentrations of total protein, as measured by the Bradford assay, were loaded to a NuPAGE 10% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA), and the electrophoresed proteins were transferred to an Amersham Protran Premium 0.45 µm nitrocellulose membrane (GE Healthcare 10600096). The membrane was then blocked (1% skim milk in PBS) and probed with chimeric MAb or immunized rabbit sera. Rabbit anti-SNAP-25 polyclonal antibody raised against a synthetic peptide corresponding to the N-terminus of human SNAP-25 (Sigma S9684), was used as a positive control to detect both the cleaved and the intact SNAP-25. AP-conjugated anti-human IgG (Sigma A3187) and AP-conjugated anti-rabbit IgG (Jackson 711-066-152) were used as secondary antibodies for chimeric MAb and rabbit sera, respectively. 5-Bromo-4-Chloro-3-Indolyl-phosphate (BCIP, Sigma B8503) was used as the chromogenic substrate. Densitometry analysis showed that the total SNAP-25 (cleaved and intact) in the BoNT/E treated samples produced similar signals, further confirming that equal amounts of the subject protein were loaded to the gels (Figure S1).

A standard direct ELISA protocol was applied essentially as described [18 (link)]. Microtiter plates were coated using 0.1 μg/well of recombinant SARS-CoV-2 spike, S1 domain, RBD, NTD subunits or huACE2 (Sino Biological, Beijing, China). AP-conjugated anti-rabbit IgG or AP-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied for rabbit sera or recombinant antibodies, respectively. Detection was performed using PNPP substrate (Sigma, Rehovot, Israel). Data points were fitted using non-linear regression (Prism 5, GraphPad, San Diego, CA, USA).