Figure 1.
Production of cTA12 in Sf9 cells by infection of a recombinant baculovirus at different multiplicities of infection (MOI).
1.35×107 cells seeded in T75 flasks were infected at different MOI (•: 0.04; ▪: 0.12; ▴0.4; ⧫1.2) with the recombinant baculovirus stock. cTA12 containing supernatants were harvested at different times and antibody was quantified by enzyme immunoassay.
Figure 2.
Western blot analysis of cTA12 produced in insect cells and mammalian cells.
Samples of TA12 were denatured at 95°C in Laemmli buffer and electrophoresed in 13% SDS-PAGE; in non-reducing conditions (A), lane 1: cTA12 purified from ascitic fluids (clone 16G7) and lane 2: cTA12 purified from Sf9 supernatant; and in reducing conditions (B) Lane 1: Sf9 supernatant, lane 2: cTA12 purified from Sf9 supernatant, lane 3: cTA12 purified from ascitic fluids (clone 16G7), lane 4: unpurified ascitic fluids (clone 16G7). After transfer, membranes were incubated with a polyclonal rabbit anti-human antibody, detected with HRP-conjugated anti-rabbit IgG and revealed using chemiluminescence.
Figure 3.
Productivity of different SP2/0-Ag14 clones secreting cTA12.
6-well plates were seeded with 2.5×105 cells/well in 3 ml of medium culture. The productivity of 12 clones obtained after stable transfection and limiting dilution cloning was tested +12A1; ¤12A2; ▴13E1; X 13E4; ⧫13E5; •16C2; □16C4; ▵16G1; ▾16G2; ◊16G3; ○16G6; ▪16G7). Supernatants were harvested every 24 h to measure cell density and cTA12 concentration (by enzyme immunoassay).
Figure 4.
Kinetic analyses of cTA12 and mTA12 binding to Fc-BoNT/A1 by SPR.
mTA12 was directly immobilized on a sensor chip at 200 RU (A), and cTA12 was captured at 150 RU using a sensor chip prepared with immobilized anti-human antibody (B). Fc-BoNT/A1 (range 0.8 to 20 nM) was injected at 50 µl/min for 3 min (association phase). Dissociation was monitored over a period of 30 min before the chip was regenerated with 10 mM glycine pH 2. BiaEvaluation Software 3.2 was used to calculate kinetic constants with the Langmuir 1∶1 model fit.
Table 1.
Association (kon) and dissociation (koff) rate constants and equilibrium dissociation constant (KD) of murine and chimeric TA12 binding to Fc-BoNT/A1.
Figure 5.
Quantification of functional cTA12 recognising native BoNT/A1 by a sandwich immunoassay.
Native BoNT/A1 from culture supernatant (100 µl at 300 LD100) was incubated in 96-well microtitre plates coated with different dilutions of purified TA12 (from 0.05 to 1.5 µg/ml). TA13-labeled AChE was used as the tracer antibody, and absorbance was measured at 414 nm after incubation with AChE substrate. cTA12 purified from ascitic fluids and cTA12 purified from Sf9 supernatant were compared using mTA12 as reference (□ purified mTA12; ▪ cTA12 purified from ascitic fluids; ▪ cTA12 purified from Sf9 supernatant).
Figure 6.
Pharmacokinetic analysis of cTA12 and mTA12 in mice.
50 µg of purified antibody was intraperitoneally injected into Swiss mice (n = 3 or 4). Mice were sacrificed at different times to calculate plasma concentrations of mAb (• mTA12; cTA12) using a competitive immunoassay. Data were analysed and fitted with WinNonLin professional software (Pharsight®).
Table 2.
B0NT/A1 neutralisation by the murine and the chimeric TA12 mAb according to the reference method L+10 from the Pharmacopoeia.
Table 3.
Neutralization potency of cTA12 using the in vivo co-injection protocol.
Table 4.
BoNT/A1, BoNT/A2, and BoNT/A3 protection by the murine and chimeric TA12 mAbs.