Figure 1: Characterization of the MethAb.

(a) The genetic construct of the Meth-specific monoclonal antibody (MethAb). Both heavy-chain and light-chain genes of MethAb were linked by a 2A self-cleavage sequence and driven by the cytomegalovirus (CMV) promoter in the expression vector pAAV-MethAb. Linker = a sequence composed of a furin cleavage site (RKRR), a V5 epitope, and a spacer peptide (SGSG); SP = signal peptide; WPRE = woodchuck hepatitis B virus post-transcriptional regulatory element; 6 × His = 6 × histidine tag. (b) Mouse IgG (green) immunoreactivity was found in 293 cells pretreated with AAV-MethAb, but not in naïve cells. Cellular nuclei were labeled with DAPI (blue). Calibration = 25 μm. (c) A secreted full-length antibody was generated by 293 cells transduced with AAV-MethAb. Culture media were harvested from transduced cells and subjected to antibody purification by using protein-G conjugated magnetic beads. Purified samples were separated by SDS-PAGE under reducing or nonreducing conditions and probed with an anti-mouse-IgG polyclonal antibody or an anti-6 × His polyclonal antibody in Western blot analysis. Mock = samples were purified from the culture media of un-transduced 293 cells; NeuN = a commercially available mouse mAb recognizing the neuron-specific nuclear protein. (d) The antibody, generated by AAV-MethAb transduced 293 cells, can bind to Meth-HRP. Purified MethAb was subjected to a colorimetric assay to assess its binding activity to Meth molecules. The conversion of horseradish peroxidase (HRP) substrates to blue-colored substances was measured at 450 nm. ***p < 0.0001. Anti-V5 = a mouse mAb recognizing the V5 epitope derived from the simian parainfluenza virus type 5. (e) MethAb can pull down Meth. 293 cells were transfected with pAAV-MethAb or transfection reagent only (control). The secreted MethAb was purified by His-Mag Sepharose beads at 48 h post-transfection and then incubated with 150 μl of Meth (50 ng/ml) to pull down Meth molecules. After that, beads were collected by a magnet, and the supernatant was subjected to the quantification of Meth. The amount of pulled-down Meth was calculated. (f) The purified MethAb used for the precipitation of Meth was separated by SDS-PAGE under the reducing condition. The heavy chain (HC, HC-Linker-2A) and light chain (LC) of MethAb were detected by an anti-mouse-IgG goat polyclonal antibody in Western blot analysis.