Goat anti human kappa hrp

ELISA assay plates were coated overnight at 4°C with 100 μL/well of 1 μg/mL recombinant human Trop-2-IgFc chimera protein (rhTROP-2, Cat #650-T2-100, R&D, Minneapolis, MN, United States), in 0.2 M sodium carbonate buffer (pH 9.4). Well surfaces were blocked with 300 µL/well of blocking buffer (2% skim milk in PBS, 0.05% Tween-20), for 30 min RT. The plates were washed twice with wash buffer (PBS, 0.05% Tween-20). Purified antibodies or supernatants were added to the plates at appropriate serial dilutions, from 5 to 10 μg/mL, followed by serial 3-fold dilutions, 100 µL/well. All dilutions were performed in blocking buffer. Antibody-containing plates were incubated for 1 h at RT, then washed 3 times with wash buffer. Antibody binding was revealed with 100 µL/well of a 1:2000 dilution of goat anti-human kappa-HRP (Cat # 2060-05, Southern Biotech, Birmingham, AL, United States) in blocking buffer, incubated for 30 min at RT, and followed by four washes with wash buffer. HRP activity was quantified by adding 100 μL/well ABTS substrate (AMRESCO, Solon, OH), activated with 20 μL 30% H₂O₂ per 10 mL ABTS solution. The reaction was stopped with 100 µL/well 2% oxalic acid. Absorbance was read at 405 nm.

SDS-PAGE and western blot assays were carried out to confirm the size and assembly of plant-made anti-PD-L1 mAb. Protein samples including non-infiltrated crude extract, agroinfiltrated crude extract, purified plant-produced Atezolizumab, and Tecentriq (Roche, Switzerland) were either mixed with non-reducing loading dye (125 mM Tris-HCl pH 6.8, 12% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue) or reducing loading dye (non-reducing loading dye with 22% (v/v) β-mercaptoethanol). The samples were separated in 4-15% polyacrylamide gel and visualized by InstantBlue (Abcam, Cambridge, UK). For western blot analysis, the proteins were transferred to nitrocellulose membrane (Bio-Rad, Massachusetts, USA). The membrane was blocked with 5% (w/v) skim milk in 1×PBS. Goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:5,000 and goat anti-human IgG-HRP (SouthernBiotech, Alabama, USA) at 1:10,000 in 3% (w/v) skim milk were used as detection antibodies. Blots were developed using ECL substrate (Promega, Wisconsin, USA) and exposed to X-ray film (Carestream, New York, USA).

Concentration of mAb in crude and purified samples were calculated by quantitative sandwich ELISA. A 96-well, half-area microtiter plate (Corning, New York, USA) was coated with goat anti-human IgG Fc (Abcam, Cambridge, UK) overnight at 1:1,000 dilution in 1×PBS. Plate washing was with 1×PBS-T (1×PBS with 0.05% Tween 20), and blocking was with 5% (w/v) skim milk in 1×PBS. Serially diluted plant samples and human IgG1 kappa isotype antibody standard (Abcam, Cambridge, UK) were incubated on the coated plates for 2 h at 37 °C, followed by detection with goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:3,000 in 1×PBS. Finally, TMB one solution substrate (Promega, Wisconsin, USA) was added and the reaction was quenched with 1M H₂SO₄. The absorbance was measured at 450 nm on a NS-100 Nano Scan microplate reader (Hercuvan, Shah Alam. Malaysia).