Figure 3
Generation and characterization of recombinant 2.44IgG1. (A) Expression cassette of pTRAkt_HC and pTRAkt_LC. The vectors contain the CaMV 35S promoter , a 5′ UTR of the TEV (5′ UTR (TEV)) and a murine IgG signal sequence targeting the apoplast Two vectors were constructed, one containing the genetic information for the hCγ1 sequence (pTRAkt_HC) and one containing the genetic information for the hCκ1 sequence (pTRAkt_LC). The rescued VH and VL regions were inserted using the AgeI/SalI (VH) or AgeI/BsiWI (VL) restriction sites. (B) Purity and assembly of recombinant 2.44IgG1. Recombinant 2.44IgG1 was purified from filtered plant extract by MabSelectTM chromatography, and 2 μg of purified 2.44IgG1 was analysed for purity and correct antibody assembly by SDS-PAGE (12% polyacrylamide) under non-reducing (nr) and reducing (r) conditions. (C) Recognition of mE-ERH by recombinant 2.44IgG1. The antigen-binding activity of recombinant 2.44IgG1 was analysed by ELISA using 100 ng mE-ERH per well. Recombinant 2.44IgG1 was probed with goat anti-human IgGFc PO, visualized with TMB whose reaction was stopped with 1 M HCl. (D) Competition of recombinant 2.44IgG1 by parental murine IgGs. To compare the binding activity of the recombinant antibody and the three murine antibodies, a competition ELISA was carried out using a constant concentration of 500 ng/ml recombinant 2.44IgG1 and increasing concentrations of the murine antibodies. Recombinant 2.44IgG1 was probed with goat anti-human IgGFc PO, visualized with TMB whose reaction was stopped with 1 M HCl.