Next-Gen Antibody: Transforming ADC Research Payload Antibody

Sample: SK-BR-3 cells (wild type(WT) / SLC46A3^* knockout(KO) )
＊ SLC46A3: lysosomal membrane protein
Treatment: DM1-conjugated antibody (2 μg/mL) for 15 min, washed, and incubated overnight.
Fixation: 4% PFA
Staining: Primary Ab: DM1 antibody (PP-AB12204-0R, 5 μg/mL)
       Secondary Ab: Mouse IgG-Alexa Fluor 488 (Green)
       Counterstain: Hoechst 33342 (Blue)

※ Courtesy of Prof. Katsuhisa Inoue of Tokyo University of Pharmacy and Life Sciences

Anti-DM1 antibody (PP-AB12204-0R) was immobilized at 2.5 μg/mL. Serial dilutions of DM1-Ab or Naked Ab, diluted in 50% mouse plasma, were added. This was followed by the addition of HRP-labeled anti-human IgG antibody, and detection was performed using an HRP-based immunoassay.

Limit of Detection: 0.14 ng/mL
Range of Quantification: 0.41-11.1 ng/mL

Recombinant DXd-binding protein was immobilized at 2.5 μg/mL. After blocking, serial dilutions of DXd-Ab or Naked antibody in 50% mouse plasma were added. Subsequently, HRP-labeled anti-DXd/Exatecan antibody (PP-AB12703-0R) was added at a 1:2,500 dilution (final conc.: 0.2 μg/mL), and detection was performed using an HRP-based immunoassay.

Limit of Detection:0.68 ng/mL
Range of Quantification: 2.06-55.6 ng/mL

Following a single 3 mg/kg dose in mice, plasma pharmacokinetics of DXd-Ab were measured using a bridging ELISA with recombinant DXd-Ab binding protein and HRP-labeled anti-DXd/Exatecan antibody (Mean ± SD, n=3 per time point). Blood samples were collected at 5 min and 3, 7 and 14 days post-dose. Plasma samples were diluted 1:16,000–1:128,000 for the 5-min time point, and 1:4,000–1:32,000 for the 3–14 day time points with each dilution measured in triplicate. Concentrations within the validated range were back-calculated, and the mean value of eligible dilutions was calculated for each animal.

We investigated the effect of the coating concentration of the anti-MMAE antibody. Anti-MMAE antibody (PP-AB12401-0R) was immobilized at 2.5 and 5.0 μg/mL, followed by the addition of a serial dilution of MMAE-conjugated antibodies. Subsequently, HRP-labeled anti-human IgG antibody was added, and detection was performed via HRP-based immunoassay.

Anti-MMAE antibody (PP-AB12401-0R) was immobilized at 2.5 μg/mL. A serial dilution of either MMAE-Ab or Naked Ab, diluted in 50% mouse plasma, was then added. Subsequently, HRP-labeled anti-human IgG antibody was added, and detection was performed via HRP-based immunoassay.

Limit of Detection: 1.37 ng/mL
Range of Quantification: 4.12-111 ng/mL