NeuroMab™ Anti-BACE1 BBB Shuttle Antibody, Clone 1A11

Figure 1 The inhibitory effect of mAbs 5G7, 14F10 and 1A11 on BACE1 in the BACE1 MBP-C125Swe experiment. The substrate is the fusion protein of maltose binding protein (MBP) and the carboxy-terminal 125 amino acids of human APP containing the Swedish double mutation, and the IC50 is three The mAbs were 0.47 nM (5G7), 0.46 nM (14F10) and 0.76 nM (1A11).

Figure 2 The regulation of BACE1 activity in the mcaFRET experiment, the substrate is the small molecule FRET peptide MCA-SEVENLDAEFRK(Dnp)-RRRR—NH2, the IC50 (or EC50) of the three mAbs are 0.06 nM (5G7), 1.6 nM (14F10 ) and 0.38 nm (1A11).

Figure 3 MAb 1A11 inhibits BACE1 activity in SH-SY5Y/APPwt cells. SH-SY5Y/APPwt cells were treated with 300 nM MAbs 1A11, 5G7 and 14F10 in PBS. PBS was used as negative control, while complex BACE1 inhibitor was used as positive control (CT+). After 24 hours of treatment, Aβ and sAPPβ in the conditioned medium were analyzed by Western blot, and MAb 1A11 treatment reduced the production of Aβ and sAPPβ.

Figure 4 MAb 1A11 inhibits BACE1 activity in primary cultured mouse neurons.

Primary cultured neurons were transduced with human APPwt by Semliki forest virus and treated with 100 nM MAb 1A11, MAb 5G7 and MAb 14F10 (dissolved in PBS). PBS was used as a negative control, while a BACE1 compound inhibitor was used as a positive control. After 24 hours of treatment, conditioned media and cell extracts were analyzed by Western blot. MAb 1A11 treatment strongly inhibited the production of Aβ, sAPPβ and CTFβ.

Figure 5 Dose-dependent inhibition of BACE1 activity by MAB 1A11 in primary cultured mouse neurons.

Mouse primary cultured neurons were transduced with human APPwt by Semliki forest virus and treated with MAb 1A11 dilutions ranging from 0.031 nM to 100 nM. Neurons were metabolically labeled with 355-methionine for 6 hours. Full-length APP and CTF were detected from cell extracts by fluorescence imaging after IP using a polyclonal antibody to the APP C-terminus. Aβ and sAPPβ from conditioned media were analyzed by direct western blot.

Figure 6 Dose-dependent inhibition of BACE1 activity by MAB 1A11 in primary cultured mouse neurons.

Quantification of CTFβ levels to inhibit BACE1 activity

Figure 7 Antigen-binding fragment (Fab) of MAb 1A11 inhibits BACE1 activity in primary cultured mouse neurons.

Primary cultured neurons were transduced with human APPwt by Semliki forest virus and treated with 200 nM Fabs produced by MAb 1A11, MAb 5G7 and MAb 14F10 (dissolved in PBS). After 24 hours of treatment, conditioned media and cell extracts were analyzed by Western blot. The Fab of MAb 1A11 strongly inhibits the production of Aβ, sAPPβ and CTFβ.

Figure 8 Stereotaxic administration of MAb 1A11 inhibits BACE1 activity in vivo.

Stereotaxically inject 1 μl of MAb 1A11 (4 μg/μl in PBS) into the right hemisphere hippocampus and cortex of wild-type mice. As a control, PBS was injected into the hippocampus and cortex of the left hemisphere. 24 hours after injection, mice were sacrificed and brain samples were analyzed by Western blot. MAb 1A11 inhibits CTFβ production in the hippocampus and cortex.

Figure 9 Immunofluorescence staining of HEK-BACE1 cells with MAb 1A11 and 5G7.

Cells fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 were stained.

Figure 10 Mutagenesis of amino acids 376-379 on loop F (mut376-9 SQDD to WAAA) and amino acids 332-334 on loop D (mut332-4 QAG to AGA) abolished immunoprecipitation of MAb 1A11 to BACE1 reactivity.

Wild-type and mutant forms of BACE1 were expressed in mammalian cells, and cell extracts were used for immunoprecipitation. Immunoprecipitates were detected by Western blot using anti-BACE1 monoclonal antibody 10B8. Similar amounts of wild-type and mut376-9 mutant BACE1 were used as input for immunoprecipitation.

Figure 11 Mutagenesis of amino acids 376-379 on loop F (mut376-9 SQDD to WAAA) and amino acids 332-334 on loop D (mut332-4 QAG to AGA) abolished immunoprecipitation of MAb 1A11 to BACE1 reactivity.

Wild-type and mutant forms of BACE1 were expressed in mammalian cells, and cell extracts were used for immunoprecipitation. Immunoprecipitates were detected by Western blot using anti-BACE1 monoclonal antibody 10B8. Similar amounts of wild-type and mut332-4 mutant BACE1 (Panel B) were used as input for immunoprecipitation.